Cytotoxic and anti-mitotic compounds, and methods of using the same

ABSTRACT

Compounds having cytotoxic and/or anti-mitotic activity are disclosed. The compounds have the following structure (I): 
                         
including stereoisomers, pharmaceutically acceptable salts and prodrugs thereof, wherein R 1 , R 2 , R 3 , R 4  and R 5  are as defined herein. Methods associated with preparation and use of such compounds, as well as pharmaceutical compositions comprising such compounds, are also disclosed. Also disclosed are compositions having the structure: (T)-(L)-(D), wherein (T) is a targeting moiety, (L) is an optional linker, and (D) is a compound having structure (I).

REFERENCE TO PRIOR APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/792,020, filed Mar. 15, 2013, and U.S. Provisional Application No.61/792,066, filed Mar. 15, 2013, the disclosure of each of which isincorporated by reference herein in its entirety.

BACKGROUND

Field

The invention relates to biologically active compounds, compositionscomprising the same, and methods of using such biologically activecompounds and compositions for the treatment of cancer and otherdiseases.

Description of the Related Art

Talpir, R. et al. (1994) Tetrahedron Lett. 35:4453-6, describe thenaturally occurring compound hemiasterlin, a stable tripeptide obtainedfrom marine sponges that causes microtubule depolymerization and mitoticarrest in cells. Hemisasterlin consists of unusual and highly congestedamino acids, features thought to contribute to its activity. A number ofgroups have modified particular structural elements of hemiasterlin toevaluate structure-activity relationships and assess the activity ofhemiasterlin analogs. See for example Zask et al., Bioorganic &Medicinal Chemistry Letters, 14:4353-4358, 2004; Zask et al., J MedChem, 47:4774-4786, 2004; Yamashita et al., Bioorganic & MedicinalChemistry Letters, 14:5317-5322, 2004; PCT/GB96/00942; WO 2004/026293;WO96/33211; and U.S. Pat. No. 7,579,323.

Analogs of hemiasterlin with modifications in the “A-segment”, or theamino terminal segment, have been described (see for example, Zask etal., J Med Chem, 47:4774-4786, 2004; Yamashita et al., Bioorganic &Medicinal Chemistry Letters, 14:5317-5322, 2004; U.S. Pat. No.7,579,323). U.S. Pat. No. 7,579,323 discloses an analog of hemiasterlin,referred to as HT1-286, in which the indole moiety is replaced by aphenyl group. HT1-286 exhibits potent anti-mitotic activity and has beenassessed in clinical trials for the treatment of cancer (Ratain et al.,Proc Am Soc Clin Oncol, 22:129, 2003).

Analogs of hemiasterlin with modifications in the “D-segment”, or thecarboxy terminal segment, have also been reported (see, for example, WO2004/026293; Zask et al., Bioorganic & Medicinal Chemistry Letters,14:4353-4358, 2004; Zask et al., J Med Chem, 47:4774-4786, 2004). Themajority of modifications at the carboxy terminus result in compoundswith substantially decreased potency compared to parent carboxylicacids. See, for example, WO 2004/026293, particularly Table 12. Zask etal., (J Med Chem, 47:4774-4786, 2004) also report that amide analogsprepared using simple cyclic and acyclic amines exhibit significantlyreduced potency (reductions of one to three orders of magnitude). Amongthe few tolerated modifications, Zask et al., (Bioorganic & MedicinalChemistry Letters, 14:4353-4358, 2004) report that the addition ofesterified cyclic amino acids at the carboxy-terminus yieldstetrapeptide analogs with prolyl-like ester-containing termini, some ofwhich exhibit potency comparable to parent compound in a tested cancercell line.

Potent cytotoxic and anti-mitotic compositions are highly desired forthe treatment of a number of devastating disorders, including cancer.While a wide variety of hemiasterlin analogs have been generated, many,including a wide variety of compounds with modifications at the carboxyterminus, exhibit reduced potency that limits utility in methods ofmedical treatment.

For the foregoing reasons, while progress has been made in this field,there is a need for additional potent anti-mitotic and cytotoxiccompounds having preferred characteristics that render them suitable forthe treatment of a variety of disorders, including cancer. The presentdisclosure fulfills these needs and provides further related advantages.

BRIEF SUMMARY

In brief, the present disclosure is directed to biologically activecompounds, compositions comprising the same, and methods of using suchcompounds and compositions.

In one embodiment, compounds having the following structure (I) areprovided:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein:

R₁ is selected from the group consisting of optionally substitutedalkyl, optionally substituted alkylamino, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedheterocyclyl and optionally substituted heteroaryl;

R₂ is selected from the group consisting of optionally substitutedalkyl, optionally substituted alkylamino, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedheterocyclyl and optionally substituted heteroaryl;

R₃ is selected from the group consisting of H and C₁₋₆alkyl;

R₄ is selected from the group consisting of H and C₁₋₆alkyl; and

R₅ is selected from the group consisting of C₁₋₆alkyl and —SH.

In one embodiment, the invention provides a compound exhibiting an EC₅₀more potent than that of HTI-286.

In one embodiment, the invention provides a method of making a compoundhaving structure (I).

In another embodiment, a pharmaceutical composition is providedcomprising a compound having structure (I), or a stereoisomer,pharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, a method of using a compound having structure (I)in therapy is provided. In particular, the present disclosure provides amethod of treating cancer in a mammal comprising administering to amammal in need thereof an effective amount of a compound havingstructure (I) or a pharmaceutical composition comprising a compoundhaving structure (I) and a pharmaceutically acceptable carrier diluentor excipient.

In another embodiment, the present disclosure provides a method ofinhibiting tumor growth in a mammal comprising administering to a mammalin need thereof an effective amount of a compound having structure (I)or a pharmaceutical composition comprising a compound having structure(I) and a pharmaceutically acceptable carrier, diluent or excipient.These and other aspects of the disclosure will be apparent uponreference to the following detailed description.

In another embodiment, the present disclosure provides a method ofkilling cancer cells in vitro using a compound having structure (I). Inanother embodiment, the present disclosure provides a method of killingcancer cells in vivo in a mammal, comprising administering to a mammalin need thereof an effective amount of a compound having structure (I)or a pharmaceutical composition comprising a compound having structure(I) and a pharmaceutically acceptable carrier, diluent or excipient.

In one embodiment, the invention provides compositions having thefollowing structure:(T)-(L)-(D)  (II)wherein (T) is a targeting moiety, (L) is an optional linker, and (D) isa compound having structure (I), or a stereoisomer, pharmaceuticallyacceptable salt or prodrug thereof. (D) is covalently attached to (L),if (L) is present, or (T), if (L) is not present.

In one embodiment, the targeting moiety is an antibody. Accordingly, inone embodiment, antibody-drug conjugates (ADCs) comprising compoundshaving structure (I), or a stereoisomer, pharmaceutically acceptablesalt or prodrug thereof, are provided.

In one embodiment, the invention provides a method of making acomposition having structure (II).

In another embodiment, a pharmaceutical composition is providedcomprising a composition having structure (II), or a stereoisomer,pharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, a method of using a composition having structure(I) in therapy is provided. In particular, the present disclosureprovides a method of treating cancer in a mammal comprisingadministering to a mammal in need thereof an effective amount of acomposition having structure (II) or a pharmaceutical compositioncomprising a composition having structure (II) and a pharmaceuticallyacceptable carrier diluent or excipient.

In another embodiment, the present disclosure provides a method ofinhibiting tumor growth in a mammal comprising administering to a mammalin need thereof an effective amount of a composition having structure(II) or a pharmaceutical composition comprising a composition havingstructure (II) and a pharmaceutically acceptable carrier, diluent orexcipient.

In another embodiment, the present disclosure provides a method ofkilling cancer cells in vitro using a composition having structure (II).In another embodiment, the present disclosure provides a method ofkilling cancer cells in vivo in a mammal, comprising administering to amammal in need thereof an effective amount of a composition havingstructure (II) or a pharmaceutical composition comprising a compositionhaving structure (II) and a pharmaceutically acceptable carrier, diluentor excipient.

These and other aspects of the disclosure will be apparent uponreference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows summarized cytotoxicity data (EC₅₀) for each of CompoundsA-E for two cell lines (HCC1954 and Jurkat).

FIG. 2 shows a cytotoxicity data plot for Compound A on two cell lines(HCC1954 and Jurkat).

FIG. 3 shows a cytotoxicity data plot for Compound B on two cell lines(HCC1954 and Jurkat).

FIG. 4 shows a cytotoxicity data plot for Compound C on two cell lines(HCC1954 and Jurkat).

FIG. 5 shows a cytotoxicity data plot for Compound D on two cell lines(HCC1954 and Jurkat).

FIG. 6 shows a cytotoxicity data plot for Compound E on two cell lines(HCC1954 and Jurkat).

FIG. 7 shows a cell kill curve on HCC 1954 cells in vitro with theantibody-drug conjugates: T-LC-SPDP-A (Trastuzumab, LC-SPDP linker,Compound A) and T-SMCC-A (Trastuzumab, SMCC linker, Compound A). EC₅₀values are shown in the figure.

FIG. 8 shows a cell kill curve on HCC 1954 cells in vitro with theantibody-drug conjugates: T-SPDP-B (Trastuzumab, LC-SPDP linker,Compound B) and T-SMCC-A (Trastuzumab, SMCC linker, Compound B). EC₅₀values are shown in the figure.

FIG. 9 shows a cell kill curve on HCC 1954 cells in vitro with theantibody-drug conjugate: T-LC-SPDP-C (Trastuzumab, LC-SPDP linker,Compound C). EC₅₀ value is shown in the figure.

FIG. 10 shows a cell kill curve on HCC 1954 cells in vitro with theantibody-drug conjugates: T-MCvcPABC-85 (Trastuzumab, MCvc PABC linker,Compound 85), T-MCvcPABC-77 (Trastuzumab, MCvc PABC linker, Compound 77)and T-MCvcPABC-80 (Trastuzumab, MCvc PABC linker, Compound 80). EC₅₀values are shown in the figure.

FIG. 11 shows a cell kill curve on BxPC-3 cells in vitro with theantibody-drug conjugate C-MCvcPABC-77, (Cetuximab, MCvc PABC linker,Compound 77), and a cell kill curve on HPAF-II cells in vitro with theantibody-drug conjugate C-MCvcPABC-77, (Cetuximab, MCvc PABC linker,Compound 77). EC₅₀ values are shown in the figure.

FIG. 12 shows a cell kill curve on HCC1954 cells in vitro with theantibody-drug conjugates: T-MCvcPABC-77, (Trastuzumab, MCvc PABC linker,Compound 77), T-MCvcPABC-85, (Trastuzumab, MCvc PABC linker, Compound85), T-MCvcPABC-58, (Trastuzumab, MCvc PABC linker, Compound 58), andT-MCvcPABC-63, (Trastuzumab, MCvc PABC linker, Compound 63). EC₅₀ valuesare shown in the figure.

FIG. 13 shows a cell kill curve on NCI-N87 cells in vitro with theantibody-drug conjugates: T-MCvcPABC-77, (Trastuzumab, MCvc PABC linker,Compound 77), T-MCvcPABC-63, (Trastuzumab, MCvc PABC linker, Compound63), T-MCvcPABC-85, (Trastuzumab, MCvc PABC linker, Compound 85),T-MCvcPABC-77, (Trastuzumab, MCvc PABC linker, Compound 77), andT-MCvcPABC-80, (Trastuzumab, MCvc PABC linker, Compound 80). EC₅₀ valuesare shown in the figure.

FIG. 14 shows the in vivo results of administration of Compound F,Compound 14, or Compound 23 on tumour volume in female athymic nude micewith established tumours.

FIG. 15 shows the in vivo results of administration of antibody-drugconjugate T-MCC-DM1 (Trastuzumab, MCC linker, maytansinoid DM1) atvaried dosages as indicated, or T-MCvcPABC-77 at varied dosages asindicated, on tumour volume in female NOD/SCID Gamma mice withestablished tumours.

FIG. 16 shows the in vivo results of administration of antibody-drugconjugate T-MCvcPABC-63 at 3 mg/kg, or T-MCvcPABC-77 at 3 mg/kg, ontumour volume in female NOD/SCID Gamma mice with established tumours.

FIG. 17 shows a cell kill curve on HCC 1954 cells in vitro with theantibody-drug conjugates: T-SPDP-140 (Trastuzumab, LC-SPDP linker,Compound 140) and T-SMCC-140 (Trastuzumab, SMCC linker, Compound 140).Compound 140 is linked through the side chain of its N-terminal aminoacid. EC₅₀ values are shown in the figure.

FIG. 18 shows a cell kill curve on HCC 1954 cells in vitro with theantibody-drug conjugates: T-SPDP-142 (Trastuzumab, LC-SPDP linker,Compound 142) and T-SMCC-142 (Trastuzumab, SMCC linker, Compound 142).Compound 142 is linked through the side chain of its N-terminal aminoacid. EC₅₀ values are shown in the figure.

FIG. 19 shows a cell kill curve on HCC1954 cells in vitro with theantibody-drug conjugates: T-MCvcPABC-58, (Trastuzumab, MCvc PABC linker,Compound 58), and T-MCvcPABC-41, (Trastuzumab, MCvc PABC linker,Compound 41), and shows a cell kill curve on NCI-N87 cells in vitro withthe antibody-drug conjugates: T-MCvcPABC-58, (Trastuzumab, MCvc PABClinker, Compound 58), and T-MCvcPABC-41, (Trastuzumab, MCvc PABC linker,Compound 41). Compound 41 is linked through the side chain of itsN-terminal amino acid. Compound 58 is linked through the side chain ofits N-terminal amino acid. EC₅₀ values are shown in the figure. EC₅₀values are shown in the figure.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings. When trade names are usedherein, applicants intend to independently include the trade nameproduct formulation, the generic drug, and the active pharmaceuticalingredient(s) of the trade name product.

“Amino” refers to the —NH₂ substituent.

“Cyano” refers to the —CN substituent.

“Hydroxy” or “hydroxyl” refers to the —OH substituent.

“Imino” refers to the ═NH substituent.

“Nitro” refers to the —NO₂ substituent.

“Oxo” refers to the ═O substituent.

“Thiol” refers to the —SH substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain substituentconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. Unless stated otherwise specifically in the specification, analkyl group may be optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a substituentgroup, consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to thesubstituent group through a single or double bond. The points ofattachment of the alkylene chain to the rest of the molecule and to thesubstituent group can be through one carbon or any two carbons withinthe chain. Unless stated otherwise specifically in the specification, analkylene chain may be optionally substituted.

“Alkoxy” refers to a substituent of the formula —OR_(a) where R_(a) isan alkyl substituent as defined above containing one to twelve carbonatoms. Unless stated otherwise specifically in the specification, analkoxy group may be optionally substituted.

“Alkylamino” refers to a substituent of the formula —NHR_(a) or—NR_(a)R_(a) where each R_(a) is, independently, an alkyl substituent asdefined above containing one to twelve carbon atoms. Unless statedotherwise specifically in the specification, an alkylamino group may beoptionally substituted.

“Thioalkyl” refers to a substituent of the formula —SR_(a) where R_(a)is an alkyl substituent as defined above containing one to twelve carbonatoms. Unless stated otherwise specifically in the specification, athioalkyl group may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system substituent comprisinghydrogen, 6 to 18 carbon atoms and at least one aromatic ring. Forpurposes of this disclosure, the aryl substituent may be a monocyclic,bicyclic, tricyclic or tetracyclic ring system, which may include fusedor bridged ring systems. Aryl substituents include, but are not limitedto, aryl substituents derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene,fluorene, as-indacene, s-indacene, indane, indene, naphthalene,phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unlessstated otherwise specifically in the specification, the term “aryl” orthe prefix “ar-” (such as in “aralkyl”) is meant to include arylsubstituents that are optionally substituted.

“Aralkyl” refers to a substituent of the formula —R_(b)—R_(c) whereR_(b) is an alkylene chain as defined above and R_(c) is one or morearyl substituents as defined above, for example, benzyl, diphenylmethyland the like. Unless stated otherwise specifically in the specification,an aralkyl group may be optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon substituent consisting solely ofcarbon and hydrogen atoms, which may include fused or bridged ringsystems, having from three to fifteen carbon atoms, preferably havingfrom three to ten carbon atoms, and which is saturated or unsaturatedand attached to the rest of the molecule by a single bond. Monocyclicsubstituents include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic substituentsinclude, for example, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwisestated specifically in the specification, a cycloalkyl group may beoptionally substituted.

“Cycloalkylalkyl” refers to a substituent of the formula —R_(b)R_(d)where R_(d) is an alkylene chain as defined above and R_(g) is acycloalkyl substituent as defined above. Unless stated otherwisespecifically in the specification, a cycloalkylalkyl group may beoptionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the disclosure. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl substituent, as defined above, that issubstituted by one or more halo substituents, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring substituent which consists of two totwelve carbon atoms and from one to six heteroatoms selected from thegroup consisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl substituent may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl substituent may be optionally oxidized;the nitrogen atom may be optionally quaternized; and the heterocyclylsubstituent may be partially or fully saturated. Examples of suchheterocyclyl substituents include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“N-heterocyclyl” refers to a heterocyclyl substituent as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl substituent to the rest of the molecule is through anitrogen atom in the heterocyclyl substituent. Unless stated otherwisespecifically in the specification, a N-heterocyclyl group may beoptionally substituted.

“Heterocyclylalkyl” refers to a substituent of the formula —R_(b)R_(e)where R_(b) is an alkylene chain as defined above and R_(e) is aheterocyclyl substituent as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl substituent at the nitrogen atom. Unless stated otherwisespecifically in the specification, a heterocyclylalkyl group may beoptionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system substituentcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and at least one aromatic ring. For purposes of this disclosure,the heteroaryl substituent may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylsubstituent may be optionally oxidized; the nitrogen atom may beoptionally quaternized. Examples include, but are not limited to,azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl,benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl,benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl,benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

“N-heteroaryl” refers to a heteroaryl substituent as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl substituent to the rest of the molecule is through anitrogen atom in the heteroaryl substituent. Unless stated otherwisespecifically in the specification, an N-heteroaryl group may beoptionally substituted.

“Heteroarylalkyl” refers to a substituent of the formula —R_(b)R_(f)where R_(b) is an alkylene chain as defined above and R_(f) is aheteroaryl substituent as defined above. Unless stated otherwisespecifically in the specification, a heteroarylalkyl group may beoptionally substituted.

The term “substituted” used herein means any of the above groups (i.e.,alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl)wherein at least one hydrogen atom is replaced by a bond to anon-hydrogen atoms such as, but not limited to: a halogen atom such asF, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups,alkoxy groups, and ester groups; a sulfur atom in groups such as thiolgroups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxidegroups; a nitrogen atom in groups such as azides, amines, amides,alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines,N-oxides, imides, and enamines; a silicon atom in groups such astrialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups,and triarylsilyl groups; and other heteroatoms in various other groups.“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced by a higher-order bond (e.g., a double- ortriple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl,and ester groups; and nitrogen in groups such as imines, oximes,hydrazones, and nitriles. For example, “substituted” includes any of theabove groups in which one or more hydrogen atoms are replaced with—NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)O_(h), —NR_(g)C(═NR_(g))NR_(g)R_(h), —NR_(g)SO₂R_(h),—OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g),—SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h). “Substituted also means anyof the above groups in which one or more hydrogen atoms are replacedwith —C(═O)R_(g), —C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g),—CH₂SO₂NR_(g)R_(h). In the foregoing, R_(g) and R_(h) are the same ordifferent and independently hydrogen, alkyl, alkoxy, alkylamino,thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl,heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl. “Substituted” further means any ofthe above groups in which one or more hydrogen atoms are replaced by abond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo,alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkylgroup. In addition, each of the foregoing substituents may also beoptionally substituted with one or more of the above substituents.

The term “protecting group,” as used herein, refers to a labile chemicalmoiety which is known in the art to protect reactive groups includingwithout limitation, hydroxyl and amino groups, against undesiredreactions during synthetic procedures. Hydroxyl and amino groups whichprotected with a protecting group are referred to herein as “protectedhydroxyl groups” and “protected amino groups”, respectively. Protectinggroups are typically used selectively and/or orthogonally to protectsites during reactions at other reactive sites and can then be removedto leave the unprotected group as is or available for further reactions.Protecting groups as known in the art are described generally in Greeneand Wuts, Protective Groups in Organic Synthesis, 3rd edition, JohnWiley & Sons, New York (1999). Groups can be selectively incorporatedinto compounds of the present disclosure as precursors. For example anamino group can be placed into a compound of the disclosure as an azidogroup that can be chemically converted to the amino group at a desiredpoint in the synthesis. Generally, groups are protected or present as aprecursor that will be inert to reactions that modify other areas of theparent molecule for conversion into their final groups at an appropriatetime. Further representative protecting or precursor groups arediscussed in Agrawal, et al., Protocols for Oligonucleotide Conjugates,Eds, Humana Press; New Jersey, 1994; Vol. 26 pp. 1-72. Examples of“hydroxyl protecting groups” include, but are not limited to, t-butyl,t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl (TBDPS), triphenylsilyl,benzoylformate, acetate, chloroacetate, trichloroacetate,tri-fluoroacetate, pivaloate, benzoate, p-phenylbenzoate,9-fluorenylmethyl carbonate, mesylate and tosylate. Examples of “aminoprotecting groups” include, but are not limited to, carbamate-protectinggroups, such as 2-trimethylsilylethoxycarbonyl (Teoc),1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC),allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), andbenzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imineand cyclic imide protecting groups, such as phthalimido anddithiasuccinoyl.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the disclosure. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the disclosure that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the disclosure. In one embodiment, a prodrug is rapidlytransformed in vivo to yield the parent compound of the disclosure, forexample, by hydrolysis in blood. In one embodiment, a prodrug may bestable in plasma or blood. In one embodiment, a prodrug may be targetedform of a compound of the invention. The prodrug compound often offersadvantages of solubility, tissue compatibility or delayed release in amammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp.7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is providedin Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, Ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987.

The term “prodrug” is meant to include any covalently bonded carriers,which release the active compound of the disclosure in vivo when suchprodrug is administered to a mammalian subject. Conjugates, includingADCs, as disclosed herein, are such prodrugs of compositions havingstructure (I). Prodrugs of a compound of the disclosure may be preparedby modifying functional groups present in the compound of the disclosurein such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound of the disclosure.Prodrugs include compounds of the disclosure wherein a hydroxy, amino ormercapto group is bonded to any group that, when the prodrug of thecompound of the disclosure is administered to a mammalian subject,cleaves to form a free hydroxy, free amino or free mercapto group,respectively. Examples of prodrugs include, but are not limited to,acetate, formate and benzoate derivatives of alcohol or amidederivatives of amine functional groups in the compounds of thedisclosure and the like.

The present disclosure also meant to encompass all pharmaceuticallyacceptable compounds of structure (I) being isotopically-labelled byhaving one or more atoms replaced by an atom having a different atomicmass or mass number. Examples of isotopes that can be incorporated intothe disclosed compounds include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H,¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I,and ¹²⁵I, respectively. These radiolabelled compounds could be useful tohelp determine or measure the effectiveness of the compounds, bycharacterizing, for example, the site or mode of action, or bindingaffinity to pharmacologically important site of action. Certainisotopically-labelled compounds of structure (I), for example, thoseincorporating a radioactive isotope, are useful in drug and/or substratetissue distribution studies. The radioactive isotopes tritium, i.e. ³H,and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose inview of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed in the Preparations and Examples as set out below using anappropriate isotopically-labeled reagent in place of the non-labeledreagent previously employed.

The present disclosure is also meant to encompass the in vivo metabolicproducts of the disclosed compounds. Such products may result from, forexample, the oxidation, reduction, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the present disclosure includescompounds produced by a process comprising administering a compound ofthis disclosure to a mammal for a period of time sufficient to yield ametabolic product thereof. Such products are typically identified byadministering a radiolabelled compound of the disclosure in a detectabledose to an animal, such as rat, mouse, guinea pig, monkey, or to human,allowing sufficient time for metabolism to occur, and isolating itsconversion products from the urine, blood or other biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity. The term “antibody” refers to afull-length immunoglobulin molecule or a functionally active portion ofa full-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof. The immunoglobulin disclosed hereincan be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulinmolecule. The immunoglobulins can be derived from any species. In oneaspect the immunoglobulin is of human, murine, or rabbit origin. Inanother aspect, the antibodies are polyclonal, monoclonal,multi-specific (e.g., bispecific), human, humanized or chimericantibodies, linear antibodies, single chain antibodies, diabodies,maxibodies, minibodies, Fv, Fab fragments, F(ab′) fragments, F(ab′)₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-bindingfragments of any of the above which immunospecifically bind to a targetantigen.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855).Monoclonal antibodies also include humanized antibodies may contain acompletely human constant region and a CDRs from a nonhuman source.

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (CL) and heavy chainconstant domains, C_(H1), C_(H2) and C_(H3). The constant domains may benative sequence constant domains (e.g., human native sequence constantdomains) or amino acid sequence variant thereof.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; maxibodies; minibodies; and multispecific antibodies formedfrom antibody fragment(s).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody “which binds” an antigen of interest is one capable ofbinding that antigen with sufficient affinity such that the antibody isuseful in targeting a cell expressing the antigen.

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide derived from nature. Such native sequencepolypeptides can be isolated from nature or can be produced byrecombinant or synthetic means. Thus, a native sequence polypeptide canhave the amino acid sequence of naturally-occurring human polypeptide,murine polypeptide, or polypeptide from any other mammalian species.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on a composition of theinvention (e.g., an antibody drug conjugate (ADC)). The metabolicprocess or reaction may be an enzymatic process such as proteolyticcleavage of a peptide linker of the subject composition, or hydrolysisof a functional group such as a hydrazone, ester, or amide within thesubject composition. In the context of conjugates, including ADCs,intracellular metabolites include, but are not limited to, antibodiesand free drug which have been separated intracellularly, i.e., afterentry, diffusion, uptake or transport into a cell (e.g., by enzymaticcleavage of an ADC by an intracellular enzyme).

In the context of conjugates, including ADCs, the terms “intracellularlycleaved” and “intracellular cleavage” refer to metabolic processes orreactions inside a cell on a composition of the invention whereby thecovalent attachment, e.g., the linker (L), between the drug moiety (D)and the targeting moiety (T) (e.g., an antibody) is broken, resulting inthe free drug dissociated from (T) inside the cell. The cleaved moietiesof the subject compositions are thus intracellular metabolites (e.g., T,T-L fragment, D-L fragment, D). Accordingly, in one embodiment, theinvention provides compositions that are cleavage products of acomposition having structure (II), which cleavage products includecompositions comprising structure (I).

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl substituent may or may not be substituted and that the descriptionincludes both substituted aryl substituents and aryl substituents havingno substitution.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration (or other similar regulatory agencyof another jurisdiction) as being acceptable for use in humans ordomestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of thedisclosure. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the disclosurewith one or more molecules of solvent. The solvent may be water, inwhich case the solvate may be a hydrate. Alternatively, the solvent maybe an organic solvent. Thus, the compounds of the present disclosure mayexist as a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the disclosure may be truesolvates, while in other cases, the compound of the disclosure maymerely retain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe disclosure and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

Non-limiting examples of disorders to be treated herein include benignand malignant tumors; leukemia and lymphoid malignancies, in particularbreast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, thyroid, pancreatic, prostate or bladder cancer; neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the disclosure which, when administered to amammal, preferably a human, is sufficient to effect treatment, asdefined below, of cancer or tumour cells in the mammal, preferably ahuman. The amount of a compound of the disclosure which constitutes a“therapeutically effective amount” will vary depending on the compound,the condition and its severity, the manner of administration, and theage of the mammal to be treated, but can be determined routinely by oneof ordinary skill in the art having regard to his own knowledge and tothis disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition.

The therapeutically effective amount of the drug may reduce the numberof cancer cells; reduce the tumor size; inhibit (i.e., slow to someextent and preferably stop) cancer cell infiltration into peripheralorgans; inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent the drug may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. Compounds of the presentinvention are preferably cytotoxic. For cancer therapy, efficacy can,for example, be measured by assessing the time to disease progression(TTP) and/or determining the response rate (RR).

An “effective amount” of drug when referred to in respect of the killingof cancer cells, refers to an amount of drug sufficient to produce thekilling effect.

Solid tumors contemplated for treatment using the presently disclosedcompounds include but are not limited to: sarcoma, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer,kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovariancancer, prostate cancer, esophageal cancer, stomach cancer (e.g.,gastrointestinal cancer), oral cancer, nasal cancer, throat cancer,squamous cell carcinoma (e.g., of the lung), basal cell carcinoma,adenocarcinoma (e.g., of the lung), sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer,testicular cancer, small cell lung carcinoma, bladder carcinoma, lungcancer, non-small cell lung cancer, epithelial carcinoma, glioma,glioblastoma, multiforme astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, skin cancer, melanoma,neuroblastoma, and retinoblastoma. Blood-borne cancers contemplated fortreatment using the presently disclosed compounds include but are notlimited to: acute lymphoblastic leukemia “ALL”, acute lymphoblasticB-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblasticleukemia “AML”, acute promyelocytic leukemia “APL”, acute monoblasticleukemia, acute erythroleukemic leukemia, acute megakaryoblasticleukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia,acute undifferentiated leukemia, chronic myelocytic leukemia “CML”,chronic lymphocytic leukemia “CLL”, hairy cell leukemia, and multiplemyeloma. Acute and chronic leukemias contemplated for treatment usingthe presently disclosed compounds include but are not limited to:lymphoblastic, myelogenous, lymphocytic, and myelocytic leukemias.Lymphomas contemplated for treatment using the presently disclosedcompounds include but are not limited to: Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and polycythemia vera. Othercancers contemplated for treatment using the presently disclosedcompounds include but are not limited to: peritoneal cancer,hepatocellular cancer, hepatoma, salivary cancer, vulval cancer,thyroid, penile cancer, anal cancer, head and neck cancer, renal cellcarcinoma, acute anaplastic large cell carcinoma, and cutaneousanaplastic large cell carcinoma.

Cancers, including, but not limited to, a tumor, metastasis, or otherdisease or disorder characterized by uncontrolled or undesired cellgrowth, can be treated or prevented by administration of the presentlydisclosed compounds.

In other embodiments, methods for treating or preventing cancer areprovided, including administering to a patient in need thereof aneffective amount of a compound disclosed herein in combination with a anadditional method of treatment. In one embodiment, the additional methodof treatment includes treatment with a chemotherapeutic agent. In oneembodiment the chemotherapeutic agent is that with which treatment ofthe cancer has not been found to be refractory. In another embodiment,the chemotherapeutic agent is that with which the treatment of cancerhas been found to be refractory. The compound of the invention may beadministered before, after, or at the same time as the chemotherapeuticagent.

In one embodiment, the additional method of treatment is radiationtherapy. The compound of the invention may be administered before,after, or at the same time as the radiation.

Compounds of the invention may also be administered to a patient thathas undergone or will undergo surgery as treatment for the cancer.

In a specific embodiment, the compound of the invention is administeredconcurrently with the chemotherapeutic agent or with radiation therapy.In another specific embodiment, the chemotherapeutic agent or radiationtherapy is administered prior or subsequent to administration ofcompound of the invention, in one aspect at least an hour, five hours,12 hours, a day, a week, a month, in further aspects several months(e.g., up to three months), prior or subsequent to administration of acompound of the invention.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents listed herein orotherwise known in the art can be administered. With respect toradiation, any radiation therapy protocol can be used depending upon thetype of cancer to be treated. For example, but not by way of limitation,x-ray radiation can be administered; in particular, high-energymegavoltage (radiation of greater that 1 MeV energy) can be used fordeep tumors, and electron beam and orthovoltage x-ray radiation can beused for skin cancers. Gamma-ray emitting radioisotopes, such asradioactive isotopes of radium, cobalt and other elements, can also beadministered.

Additionally, methods of treatment of cancer with a compound of theinvention are provided as an alternative to chemotherapy or radiationtherapy where the chemotherapy or the radiation therapy has proven orcan prove too toxic, e.g., results in unacceptable or unbearable sideeffects, for the subject being treated. Additionally, methods oftreatment of cancer with a compound of the invention are provided as analternative to surgery where the surgery has proven or can proveunacceptable or unbearable for the subject being treated.

The compound of the invention can also be used in an in vitro or ex vivofashion, such as for the treatment of certain cancers, including, butnot limited to leukemias and lymphomas, such treatment involvingautologous stem cell transplants. This can involve a multi-step processin which the animal's autologous hematopoietic stem cells are harvestedand purged of all cancer cells, the animal's remaining bone-marrow cellpopulation is then eradicated via the administration of a high dose of acompound of the invention with or without accompanying high doseradiation therapy, and the stem cell graft is infused back into theanimal. Supportive care is then provided while bone marrow function isrestored and the animal recovers.

Methods for treating cancer further include administering to a patientin need thereof an effective amount of a compound of the invention andanother therapeutic agent that is an anti-cancer agent. Suitableanticancer agents include, but are not limited to, methotrexate, taxol,L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine,cyclophosphamide, Ifosfamide, nitrosoureas, cisplatin, carboplatin,mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustards,cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins,bleomycin, doxorubicin, idarubicin, daunorubicin, actinomycin D,dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,vincristine, vindesine, vinorelbine, paclitaxel, and docetaxel.

Other examples of chemotherapeutic agents include alkylating agents suchas thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such asbusulfan, treosulfan, improsulfan and piposulfan; aziridines such asbenzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especiallybullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acamptothecin (including the synthetic analogue topotecan (HYCAMTIN®),CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, and uracil mustard; triazines such as decarbazine;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; epipodophyllins, such as etoposide,teniposide, topotecan, 9-aminocamptothecin, camptothecin orcrisnatol;bisphosphonates, such as clodronate; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gamma11 andcalicheamicin omega11 (see, e.g., Agnew, Chem. Intl. Ed. Engl.,33:183-186 (1994)) and anthracyclines such as annamycin, AD 32,alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100,idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, anesperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antibiotic chromophores, aclacinomysins, actinomycin,authramycin, azaserine, bleomycins (e.g., A2 and B2), cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin),esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; photodynamic therapies, such asvertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, anddemethoxy-hypocrellin A (2BA-2-DMHA); folic acid analogues such asdenopterin, pteropterin, and trimetrexate; dpurine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, cytosine arabinoside, dideoxyuridine, doxifluridine,enocitabine, and floxuridine; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, and testolactone;anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folicacid replenisher such as folinic acid (leucovorin); aceglatone;anti-folate anti-neoplastic agents such as ALIMTA®, LY231514 pemetrexed,dihydrofolate reductase inhibitors such as methotrexate andtrimetrexate; anti-metabolites such as 5-fluorouracil (5-FU) and itsprodrugs such as UFT, S-1 and capecitabine, floxuridine, doxifluridineand ratitrexed; and thymidylate synthase inhibitors and glycinamideribonucleotide formyltranferase inhibitors such as raltitrexed(TOMUDEX®, TDX); inhibitors of dihydropyrimidine dehydrogenase such aseniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids and taxanes,e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton,N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticleformulation of paclitaxel (American Pharmaceutical Partners, Schaumberg,Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France);chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine;platinum; platinum analogs or platinum-based analogs such as cisplatin,oxaliplatin and carboplatin; vinblastine (VELBAN®); etoposide (VP-16);ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinca alkaloid;vinorelbine (NAVELBINE®); velcade; revlimid; thalidomide; IMiD3;lovastatin; verapamil; thapsigargin; 1-methyl-4-phenylpyridinium; cellcycle inhibitors such as staurosporine; novantrone; edatrexate;daunomycin; mtoxantrone; aminopterin; xeloda; ibandronate; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); vitamin D3 analogs,such as EB 1089, CB 1093 and KH 1060; retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovorin.

Anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®tamoxifen), raloxifene, megastrol, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene;aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN®exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA®letrozole, and ARIMIDEX® anastrozole; and anti-androgens such asflutamide, bicalutamide, nilutamide, bicalutamide, leuprolide, andgoserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosineanalog); antisense oligonucleotides, particularly those that inhibitexpression of genes in signaling pathways implicated in abherant cellproliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

The compounds of the disclosure, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present disclosure is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (+), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centres of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present disclosure contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present disclosure includestautomers of any said compounds.

As noted above, in one embodiment of the present disclosure,compositions having cytotoxic activity and/or anti-mitotic activity areprovided, the compositions having structure (I), below. In a furtherembodiment, the subject compositions exhibit cytotoxic activity againstcancer cells.

Novel Compounds

As noted above, in one embodiment of the present disclosure, compoundshaving the following structure (I) are provided:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein:

R₁ is selected from the group consisting of optionally substitutedalkyl, optionally substituted alkylamino, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedheterocyclyl and optionally substituted heteroaryl;

R₂ is selected from the group consisting of optionally substitutedalkyl, optionally substituted alkylamino, optionally substitutedcycloalkyl, optionally substituted aryl, optionally substitutedheterocyclyl and optionally substituted heteroaryl;

R₃ is selected from the group consisting of H and C₁₋₆alkyl;

R₄ is selected from the group consisting of H and C₁₋₆alkyl; and

R₅ is selected from the group consisting of C₁₋₆alkyl and —SH.

In a further embodiment, each optionally substituted alkyl, optionallysubstituted alkylamino, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heterocyclyl and optionallysubstituted heteroaryl is, independently, optionally substituted with═O, ═S, —OH, —OR₆, —O₂CR₆, —SH, —SR₆, —SOCR₆, —NH₂, —N₃, —NHR₆, —N(R₆)₂,—NHCOR₆, —NR₆COR₆, —I, —Br, —Cl, —F, —CN, —CO₂H, —CO₂R₆, —CHO, —COR₆,—CONH₂, —CONHR₆, —CON(R₆)₂, —COSH, —COSR₆, —NO₂, —SO₃H, —SOR₆ or —SO₂R₆,wherein each R₆ is, independently, alkyl optionally substituted withhalogen, —OH or —SH.

In another further embodiment, each optionally substituted aryl andoptionally substituted heteroaryl is, independently, selected from thegroup consisting of optionally substituted phenyl, optionallysubstituted naphthyl, optionally substituted anthracyl, optionallysubstituted phenanthryl, optionally substituted furyl, optionallysubstituted pyrrolyl, optionally substituted thiophenyl, optionallysubstituted benzofuryl, optionally substituted benzothiophenyl,optionally substituted quinolinyl, optionally substituted isoquinolinyl,optionally substituted imidazolyl, optionally substituted thiazolyl,optionally substituted oxazolyl, and optionally substituted pyridinyl.

In another further embodiment, R₂ is selected from one of the followingstructures (III), (IV), (V), (VI):

wherein:

-   -   Q is CR₇ or N;    -   Z is C(R₇)₂, NR₇, S, or O;    -   each R₇ is, independently, selected from the group consisting of        H, —OH, —OR₆, —O₂CR₆, —SH, —SR₆, —SOCR₆, —NH₂, —N₃, —NHR₆,        —N(R₆)₂, —NHCOR₆, —NR₆COR₆, —I, —Br, —Cl, —F, —CN, —CO₂H,        —CO₂R₆, —CHO, —COR₆, —CONH₂, —CONHR₆, —CON(R₆)₂, —COSH, —COSR₆,        —NO₂, —SO₃H, —SOR₆ or —SO₂R₆, wherein each R₆ is, independently,        alkyl optionally substituted with halogen, —OH or —SH.

In another further embodiment, R₂ is selected from the group consistingof:

In another further embodiment, R₂ is:

In another further embodiment, R₃, R₄ and R₅ are each methyl.

In another further embodiment, R₃ is H, R₄ is methyl, and R₅ is methyl.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific substituent set forth herein for aR₁, R₂, R₃, R₄, R₅, R₆ and R₇ group in the compounds of structure (I),as set forth above, may be independently combined with other embodimentsand/or substituents of compounds of structure (I) to form embodiments ofthe present disclosure not specifically set forth above. In addition, inthe event that a list of substitutents is listed for any particular R₁,R₂, R₃, R₄, R₅, R₆ and R₇ in a particular embodiment and/or claim, it isunderstood that each individual substituent may be deleted from theparticular embodiment and/or claim and that the remaining list ofsubstituents will be considered to be within the scope of the presentdisclosure.

In one embodiment, the invention provides a method of making a compoundhaving structure (I).

Conjugates Comprising Novel Compounds

Compounds having structure (I) may be used to form conjugates, forexample antibody-drug conjugates (ADCs). Accordingly, in one embodimentof the present disclosure, conjugate compositions having the followingstructure are provided:(T)-(L)-(D)  (II)wherein (T) is a targeting moiety, (L) is an optional linker, and (D) isa compound having structure (I). In one embodiment, (T) is an antibody.Accordingly, in one embodiment, antibody-drug conjugates (ADCs)comprising compounds (D) having structure (I) are provided.

As will be appreciated by the artisan of reasonable skill, a widevariety of means are available to covalently link (T)-(L)-(D). Any knownmethod may be used to link the conjugate components. Any known linkertechnology may be used to link (T) to (D). Further, (T), (L), and (D)may be modified in any suitable manner, as recognized by the artisan ofreasonable skill, in order to facilitate conjugate formation.

Targeting Moiety (T)

The Targeting moiety (T) of the subject compositions includes within itsscope any unit of a (T) that binds or reactively associates or complexeswith a receptor, antigen or other receptive moiety associated with agiven target-cell population. A (T) is a molecule that binds to,complexes with, or reacts with a moiety of a cell population sought tobe targeted. In one aspect, the (T) acts to deliver the Drug (D) to theparticular target cell population with which the (T) reacts. Such (T)sinclude, but are not limited to, large molecular weight proteins suchas, for example, full-length antibodies, antibody fragments, smallermolecular weight proteins, polypeptide or peptides, lectins,glycoproteins, non-peptides, vitamins, nutrient-transport molecules(such as, but not limited to, transferrin), or any other cell bindingmolecule or substance.

A (T) can form a bond to a Linker unit (L) or a Drug (D). A (T) can forma bond to a (L) unit via a heteroatom of the (T). Heteroatoms that maybe present on a (T) include sulfur (in one embodiment, from a sulfhydrylgroup of a (T)), oxygen (in one embodiment, from a carbonyl, carboxyl orhydroxyl group of a (T)) and nitrogen (in one embodiment, from a primaryor secondary amino group of a (T)). These heteroatoms can be present onthe (T) in the (T)'s natural state, for example a naturally-occurringantibody, or can be introduced into the (T) via chemical modification.

In one embodiment, a (T) has a sulfhydryl group and the (T) bonds to the(L) via the sulfhydryl group's sulfur atom. In another embodiment, the(T) has one or more lysine residues that can be chemically modified tointroduce one or more sulfhydryl groups. The (T) bonds to the (L) unitvia the sulfhydryl group. Reagents that can be used to modify lysinesinclude, but are not limited to, N-succinimidyl S-acetylthioacetate(SATA) and 2-Iminothiolane hydrochloride (Traut's Reagent).

In another embodiment, the (L) can have one or more carbohydrate groupsthat can be chemically modified to have one or more sulfhydryl groups.The (T) bonds to the (L) via the sulfhydryl group's sulfur atom. In yetanother embodiment, the (T) can have one or more carbohydrate groupsthat can be oxidized to provide an aldehyde (—CHO) group (see, e.g.,Laguzza et al., 1989, J. Med. Chem. 32(3):548-55). The correspondingaldehyde can form a bond with a reactive site on a portion of a (L).Reactive sites that can react with a carbonyl group on a (T) include,but are not limited to, hydrazine and hydroxylamine. Other protocols forthe modification of proteins for the attachment or association of (D)are described in Coligan et al., Current Protocols in Protein Science,vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

The (T) can include, for example a protein, polypeptide, or peptideinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derivedgrowth factor, IL-2, IL-6, transforming growth factor (“TGF”), such asTGF-α or TGF-β, vaccinia growth factor (“VGF”), insulin and insulin-likegrowth factors I and II, lectins and apoprotein from low densitylipoprotein.

The (T) can also include an antibody, such as polyclonal antibodies ormonoclonal antibodies. The antibody can be directed to a particularantigenic determinant, including for example, a cancer cell antigen, aviral antigen, a microbial antigen, a protein, a peptide, acarbohydrate, a chemical, nucleic acid, or fragments thereof. Methods ofproducing polyclonal antibodies are known in the art. A monoclonalantibody (mAb) to an antigen-of-interest can be prepared by using anytechnique known in the art. These include, but are not limited to, thehybridoma technique originally described by Kohler and Milstein (1975,Nature 256, 495-497), the human B cell hybridoma technique (Kozbor etal., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Coleet al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). The Selected Lymphocyte Antibody Method (SLAM)(Babcook, J. S., et al., A novel strategy for generating monoclonalantibodies from single, isolated lymphocytes producing antibodies ofdefined specificities. Proc Natl Acad Sci USA, 1996. 93 (15): p.7843-8.) and (McLean G R, Olsen O A, Watt I N, Rathanaswami P, Leslie KB, Babcook J S, Schrader J W. Recognition of human cytomegalovirus byhuman primary immunoglobulins identifies an innate foundation to anadaptive immune response. J Immunol. 2005 Apr. 15; 174(8):4768-78. Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, and IgD and any subclass thereof. The hybridoma producing the mAbsof use in this invention may be cultivated in vitro or in vivo.

The monoclonal antibody can be, for example, a human monoclonalantibody, a humanized monoclonal antibody, an antibody fragment, or achimeric antibody (e.g., a human-mouse antibody). Human monoclonalantibodies may be made by any of numerous techniques known in the art(e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80:7308-7312;Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982,Meth. Enzymol. 92:3-16). See also, Huse et al., 1989, Science246:1275-1281 and McLean et al. J Immunol. 2005 Apr. 15; 174(8):4768-78.

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art. Traditional production offull-length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (see, e.g., Milstein et al., 1983, Nature305:537-539; International Publication No. WO 93/08829, Traunecker etal., 1991, EMBO J. 10:3655-3659.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H2), and C_(H3) regions. It is preferred tohave the first heavy-chain constant region (C_(H1)) containing the sitenecessary for light chain binding, present in at least one of thefusions. Nucleic acids with sequences encoding the immunoglobulin heavychain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

For example, the bispecific antibodies can have a hybrid immunoglobulinheavy chain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. This asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation (International Publication No. WO 94/04690)which is incorporated herein by reference in its entirety.

For further details for generating bispecific antibodies see, forexample, Suresh et al., 1986, Methods in Enzymology 121:210; Rodrigueset al., 1993, J. Immunology 151:6954-6961; Carter et al., 1992,Bio/Technology 10:163-167; Carter et al., 1995, J. Hematotherapy4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681. Usingsuch techniques, bispecific antibodies can be prepared for use in thetreatment or prevention of disease as defined herein.

Bifunctional antibodies are also described in European PatentPublication No. EPA 0 105 360. As disclosed in this reference, hybrid orbifunctional antibodies can be derived either biologically, i.e., bycell fusion techniques, or chemically, especially with cross-linkingagents or disulfide-bridge forming reagents, and may comprise wholeantibodies or fragments thereof. Methods for obtaining such hybridantibodies are disclosed for example, in International Publication WO83/03679, and European Patent Publication No. EPA 0 217 577, both ofwhich are incorporated herein by reference.

The antibody also can be a functionally active fragment, derivative oranalog of an antibody that immunospecifically binds to a target antigen(e.g., a cancer antigen, a viral antigen, a microbial antigen, or otherantibodies bound to cells or matrix). In this regard, “functionallyactive” means that the fragment, derivative or analog is able torecognize the same antigen that the antibody from which the fragment,derivative or analog is derived recognized. Specifically, in anexemplary embodiment the antigenicity of the idiotype of theimmunoglobulin molecule can be enhanced by deletion of framework and CDRsequences that are C-terminal to the CDR sequence that specificallyrecognizes the antigen. To determine which CDR sequences bind theantigen, synthetic peptides containing the CDR sequences can be used inbinding assays with the antigen by any binding assay method known in theart (e.g., the BIA core assay) (see, e.g., Kabat et al., 1991, Sequencesof Proteins of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md.; Kabat et al., 1980, J. Immunology125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, Fab fragments, Fab′, Fv fragments andheavy chain and light chain dimers of antibodies, or any minimalfragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., asdescribed in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54).

Recombinant antibodies, such as chimeric and humanized monoclonalantibodies, comprising both human and non-human portions, which can bemade using standard recombinant DNA techniques, also can be used. (See,e.g., U.S. Pat. No. 4,816,567; and U.S. Pat. No. 4,816,397.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., U.S. Pat. No. 5,585,089.) Chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in International PublicationNo. WO 87/02671; European Patent Publication No. 0 184 187; EuropeanPatent Publication No. 0 171 496; European Patent Publication No. 0 173494; International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Publication No. 012 023; Berter et al., 1988, Science240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; Shaw etal., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat. No.5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.,1988, Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060.

Completely human antibodies can be used. Human antibodies can beprepared, for example, using transgenic mice that are incapable ofexpressing endogenous immunoglobulin heavy and light chains genes, butwhich can express human heavy and light chain genes. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies. see, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; and 5,545,806.

Human antibodies that recognize a selected epitope also can be generatedusing a technique referred to as “guided selection.” In this approach aselected non-human monoclonal antibody, e.g., a mouse antibody, is usedto guide the selection of a completely human antibody recognizing thesame epitope. (See, e.g., Jespers et al., 1994, Biotechnology12:899-903.) Human antibodies can also be produced using varioustechniques known in the art, including phage display libraries (see,e.g., Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al.,1991, J. Mol. Biol. 222:581; Quan and Carter, 2002, “The rise ofmonoclonal antibodies as therapeutics,” in Anti-IgE and AllergicDisease, Jardieu, P. M. and Fick Jr., R. B, eds., Marcel Dekker, NewYork, N.Y., Chapter 20, pp. 427-469).

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof. For example, an antibody canbe fused via a covalent bond (e.g., a peptide bond) at either theN-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, such as at least a 10, 20 or 50 amino acidportion of the protein) that is not the antibody.

Antibodies also include analogs and derivatives that are eithermodified, i.e., by the covalent attachment of any type of molecule aslong as such covalent attachment permits the antibody to retain itsantigen binding immunospecificity. For example, but not by way oflimitation, the derivatives and analogs of the antibodies include thosethat have been further modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularantibody unit or other protein, etc. Any of numerous chemicalmodifications can be carried out by known techniques, including but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis in the presence of tunicamycin, etc. Additionally,the analog or derivative can contain one or more unnatural amino acids.

The antibodies can have modifications (e.g., substitutions, deletions oradditions) in amino acid residues that interact with Fc receptors. Inparticular, antibodies include antibodies having modifications in aminoacid residues identified as involved in the interaction between theanti-Fc domain and the FcRn receptor (see, e.g., InternationalPublication No. WO 97/34631, which is incorporated herein by referencein its entirety). Antibodies immunospecific for a target antigen can beobtained commercially or other source or produced by any method known toone of skill in the art such as, e.g., chemical synthesis or recombinantexpression techniques. The nucleotide sequence encoding antibodiesimmunospecific for a cancer cell antigen can be obtained, e.g., from theGenBank database or a database like it, the literature publications, orby routine cloning and sequencing.

Examples of antibodies available for the treatment of cancer include,but are not limited to, humanized anti HER2 monoclonal antibody,HERCEPTIN® (trastuzumab; Genentech); RITUXAN® (rituximab; Genentech)which is a chimeric anti CD20 monoclonal antibody for the treatment ofpatients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA)which is a murine antibody for the treatment of ovarian cancer; Panorex(Glaxo Wellcome, NC) which is a murine IgG2a antibody for the treatmentof colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY) whichis an anti-EGFR IgG chimeric antibody for the treatment of epidermalgrowth factor positive cancers, such as head and neck cancer; Vitaxin(MedImmune, Inc., MD) which is a humanized antibody for the treatment ofsarcoma; Campath I/H (Leukosite, Mass.) which is a humanized IgG1antibody for the treatment of chronic lymphocytic leukemia (CLL); SmartMI95 (Protein Design Labs, Inc., CA) which is a humanized anti-CD33 IgGantibody for the treatment of acute myeloid leukemia (AML); LymphoCide(Immunomedics, Inc., NJ) which is a humanized anti-CD22 IgG antibody forthe treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein DesignLabs, Inc., CA) which is a humanized anti-HLA-DR antibody for thetreatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA)which is a radiolabeled murine anti-HLA-Dr10 antibody for the treatmentof non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is ahumanized anti-CD2 mAb for the treatment of Hodgkin's Disease ornon-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA) which is ananti-VEGF humanized antibody for the treatment of lung and colorectalcancers; Epratuzamab (Immunomedics, Inc., NJ and Amgen, Calif.) which isan anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; andCEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody forthe treatment of colorectal cancer.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens (exemplary cancersare indicated in parentheses): CA125 (ovarian), CA15-3 (carcinomas),CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X(carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal),placental alkaline phosphatase (carcinomas), prostate specific membraneantigen (prostate), prostatic acid phosphatase (prostate), epidermalgrowth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas),MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti transferrin receptor(carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA(colorectal), gp100 (melanoma), MARTI (melanoma), prostate specificantigen (PSA) (prostate), IL-2 receptor (T-cell leukemia and lymphomas),CD20 (non Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22(lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiplemyeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG(melanoma), and Neu oncogene product (carcinomas). Some specific, usefulantibodies include, but are not limited to, BR96 mAb (Trail et al.,1993, Science 261:212-215), BR64 (Trail et al., 1997, Cancer Research57:100-105), mAbs against the CD40 antigen, such as S2C6 mAb (Franciscoet al., 2000, Cancer Res. 60:3225-3231) and chimeric and humanizedvariants thereof, mabs against the cD33 antigen; mabs against the EphA2antigen; mAbs against the CD70 antigen, such as 1F6 mAb and 2F2 mAb andchimeric and humanized variants thereof, and mAbs against the CD30antigen, such as AC10 (Bowen et al., 1993, J. Immunol. 151:5896-5906;Wahl et al., 2002, Cancer Res. 62(13):3736-42) and chimeric andhumanized variants thereof. Many other internalizing antibodies thatbind to tumor associated antigens can be used and have been reviewed(see, e.g., Franke et al., 2000, Cancer Biother. Radiopharm. 15:459 76;Murray, 2000, Semin. Oncol. 27:64 70; Breitling et al., RecombinantAntibodies, John Wiley, and Sons, New York, 1998).

The antibody also can be an antibody that binds to an antigen that ispresent on a target cell or target cell population. For example,transmembrane polypeptides and other markers can be specificallyexpressed on the surface of one or more particular type(s) of targetcells (e.g., a cancer cell) as compared to on one or more normal (e.g.,a non-cancerous cell(s)). Often, such markers are more abundantlyexpressed on the surface of the target cells, or exhibit greaterimmunogenicity, as compared to those on the surface of the normal cells.The identification of such cell surface antigen polypeptides has givenrise to the ability to specifically target cells for destruction viaantibody-based therapies. Thus, in some embodiments, the antibodiesinclude, but are not limited to, antibodies against tumor-associatedantigens (TAA). Such tumor-associated antigens are known in the art, andcan prepared for use in generating antibodies using methods andinformation which are well known in the art.

See also EP2552957, WO/2012/116453, WO/2012/032080. See also Zybody™,http://www.zyngenia.com/technology.html. See also human heavy chain-onlyantibodies technology, http://www.crescendobiologics.com/. See alsoWO2010001251, yeast based human antibody yeast-based platformhttp://www.adimab.com/science-and-technology/technology-overview/,mAbLogix™ platform http://www.dna.com/technology, monoclonal discoveryplatform http://www.igenica.com/technology/, WO2009/157771, EP2560993,WO2013004842, WO2012166560.

Linker Moiety (L)

The subject compositions optionally further include a Linker moiety (L).(L) is a bifunctional compound which can be used to link a (D) and a (T)to form a conjugate composition, T-L-D. Such conjugates allow theselective delivery of drugs to target cells (e.g., tumor cells). (L)sinclude a divalent substituent such as an alkyldiyl, an aryldiyl, aheteroaryldiyl, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating unitsof alkyloxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g., polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

The subject compositions can be prepared using a (L) unit having areactive site for binding to the (D) and (T). In some embodiments, (L)has a reactive site which has an electrophilic group that is reactive toa nucleophilic group present on (T). Useful nucleophilic groups on (T)include but are not limited to sulfhydryl, hydroxyl and amino groups.The heteroatom of the nucleophilic group of (T) is reactive to anelectrophilic group on (L) and forms a covalent bond to (L). Usefulelectrophilic groups include, but are not limited to maleimide andhaloacetamide groups. The nucleophilic group on (T) provides aconvenient site for attachment to (L).

In another embodiment, (L) has a reactive site which has a nucleophilicgroup that is reactive to an electrophilic group present on (T). Usefulelectrophilic groups on (T) include, but are not limited to, aldehydeand ketone carbonyl groups. The heteroatom of a nucleophilic group of(L) can react with an electrophilic group on (T) and form a covalentbond to (T). Useful nucleophilic groups on (L) include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide. The electrophilic group on (T)provides a convenient site for attachment to (L).

Carboxylic acid functional groups and chloroformate functional groupsare also useful reactive sites for (L) because they can react with aminogroups of a (D) to form an amide linkage. Also useful as a reactive siteis a carbonate functional group on (L), such as but not limited top-nitrophenyl carbonate, which can react with an amino group of a (D) toform a carbamate linkage.

It will be appreciated that any linker moieties taught in the prior art,and particularly those taught for use in the context of drug delivery,may be used in the current invention. Without limiting the scope of thepreceding statement, in one embodiment, (L) comprises a linker moietydisclosed in WO 2012/113847. In another embodiment, (L) comprises alinker moiety disclosed in U.S. Pat. No. 8,288,352. In anotherembodiment, (L) comprises a linker moiety disclosed in U.S. Pat. No.5,028,697. In another embodiment, (L) comprises a linker moietydisclosed in U.S. Pat. No. 5,006,652. In another embodiment, (L)comprises a linker moiety disclosed in U.S. Pat. No. 5,094,849. Inanother embodiment, (L) comprises a linker moiety disclosed in U.S. Pat.No. 5,053,394. In another embodiment, (L) comprises a linker moietydisclosed in U.S. Pat. No. 5,122,368. In another embodiment, (L)comprises a linker moiety disclosed in U.S. Pat. No. 5,387,578. Inanother embodiment, (L) comprises a linker moiety disclosed in U.S. Pat.No. 5,547,667. In another embodiment, (L) comprises a linker moietydisclosed in U.S. Pat. No. 5,622,929. In another embodiment, (L)comprises a linker moiety disclosed in U.S. Pat. No. 5,708,146. Inanother embodiment, (L) comprises a linker moiety disclosed in U.S. Pat.No. 6,468,522. In another embodiment, (L) comprises a linker moietydisclosed in U.S. Pat. No. 6,103,236. In another embodiment, (L)comprises a linker moiety disclosed in U.S. Pat. No. 6,638,509. Inanother embodiment, (L) comprises a linker moiety disclosed in U.S. Pat.No. 6,214,345. In another embodiment, (L) comprises a linker moietydisclosed in U.S. Pat. No. 6,759,509. In another embodiment, (L)comprises a linker moiety disclosed in WO 2007/103288. In anotherembodiment, (L) comprises a linker moiety disclosed in WO 2008/083312.In another embodiment, (L) comprises a linker moiety disclosed in WO2003/068144. In another embodiment, (L) comprises a linker moietydisclosed in WO 2004/016801. In another embodiment, (L) comprises alinker moiety disclosed in WO 2009/134976. In another embodiment, (L)comprises a linker moiety disclosed in WO 2009/134952. In anotherembodiment, (L) comprises a linker moiety disclosed in WO 2009/134977.In another embodiment, (L) comprises a linker moiety disclosed in WO2002/08180. In another embodiment, (L) comprises a linker moietydisclosed in WO 2004/043493. In another embodiment, (L) comprises alinker moiety disclosed in WO 2007/018431. In another embodiment, (L)comprises a linker moiety disclosed in WO 2003/026577. In anotherembodiment, (L) comprises a linker moiety disclosed in WO 2005/077090.In another embodiment, (L) comprises a linker moiety disclosed in WO2005/082023. In another embodiment, (L) comprises a linker moietydisclosed in WO 2007/011968. In another embodiment, (L) comprises alinker moiety disclosed in WO 2007/038658. In another embodiment, (L)comprises a linker moiety disclosed in WO 2007/059404. In anotherembodiment, (L) comprises a linker moiety disclosed in WO 2006/110476.In another embodiment, (L) comprises a linker moiety disclosed in WO2005/112919. In another embodiment, (L) comprises a linker moietydisclosed in WO 2008/103693. In another embodiment, (L) comprises alinker moiety disclosed in U.S. Pat. No. 6,756,037. In anotherembodiment, (L) comprises a linker moiety disclosed in U.S. Pat. No.7,087,229. In another embodiment, (L) comprises a linker moietydisclosed in U.S. Pat. No. 7,122,189. In another embodiment, (L)comprises a linker moiety disclosed in U.S. Pat. No. 7,332,164. Inanother embodiment, (L) comprises a linker moiety disclosed in U.S. Pat.No. 5,556,623. In another embodiment, (L) comprises a linker moietydisclosed in U.S. Pat. No. 5,643,573. In another embodiment, (L)comprises a linker moiety disclosed in U.S. Pat. No. 5,665,358. Linkers(L) comprising a self-immolative component may also be used.

For example, see U.S. Pat. No. 6,214,345. An example of aself-immolative component is p-aminobenzylcarbamoyl (PABC).

Commercially available linkers may be used in the invention. Forexample, the commercially available cleavable linker sulfosuccinimidyl6-[3′(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP: ThermoPierce Cat#21650) and Non-cleavable linker succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC: Thermo PierceCat#22360) may be used, as demonstrated herein.

See also, WO2012171020, WO2010138719, the range of commerciallyavailable linkers, for example, from Concortishttp://www.concortis.com/home. See also Kim et al., BIOCONJUGATECHEMISTRY, 21 (8): 1513-1519 August 2010. See also EP2326349. See alsocopper free click chemistry linkers, Angew. Chem. Int. Ed., 2010, 49, p.9422-9425, ChemBioChem, 2011, 12, p. 1309-1312,http://www.synaffix.com/technology/.

Drug Moiety (D)

(D) is a compound having the structure (I) as described herein. It willbe recognized by the artisan of reasonable skill that compounds ofstructure (I) may be appropriately modified to facilitate a conjugationreaction with (L), or if (L) is not present, with (T), and formation ofa conjugate (T)-(L)-(D) or (T)-(D). Any point of attachment on (D) maybe used. In one embodiment, the C-terminus of (D) forms the point ofattachment in a (T)-(L)-(D) conjugate. In another embodiment, theN-terminus of (D) forms the point of attachment in a (T)-(L)-(D)conjugate. In another embodiment, a side chain of (D) forms the point ofattachment in a (T)-(L)-(D) conjugate.

Administration

For the purposes of administration, the compounds of the presentdisclosure may be administered as a raw chemical or may be formulated aspharmaceutical compositions. Pharmaceutical compositions of the presentdisclosure comprise a compound of structure (I) and a pharmaceuticallyacceptable carrier, diluent or excipient. The compound of structure (I)is present in the composition in an amount which is effective to treat aparticular disease or condition of interest—that is, in an amountsufficient to treat cancer or tumour cell growth, and preferably withacceptable toxicity to the patient. The activity of compounds ofstructure (I) can be determined by one skilled in the art, for example,as described in the Examples below. Appropriate concentrations anddosages can be readily determined by one skilled in the art.

Administration of the compounds of the disclosure, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of administration of agents for serving similar utilities. Thepharmaceutical compositions of the disclosure can be prepared bycombining a compound of the disclosure with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Typical routes of administering such pharmaceuticalcompositions include, without limitation, oral, topical, transdermal,inhalation, parenteral, sublingual, buccal, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. Pharmaceutical compositions of the disclosure areformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that will be administered to a subject or patient take theform of one or more dosage units, where for example, a tablet may be asingle dosage unit, and a container of a compound of the disclosure inaerosol form may hold a plurality of dosage units. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington: The Science andPractice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy andScience, 2000). The composition to be administered will, in any event,contain a therapeutically effective amount of a compound of thedisclosure, or a pharmaceutically acceptable salt thereof, for treatmentof a disease or condition of interest in accordance with the teachingsof this disclosure.

A pharmaceutical composition of the disclosure may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, pharmaceutical compositions ofthe present disclosure typically are either solid or liquid form, wheresemi-solid, semi-liquid, suspension and gel forms are included withinthe forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcompositions may be formulated into a powder, granule, compressedtablet, pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

Pharmaceutical compositions of the disclosure may be in the form of aliquid, for example, an elixir, syrup, solution, emulsion or suspension.The liquid may be for oral administration or for delivery by injection,as two examples. When intended for oral administration, pharmaceuticalcompositions of the disclosure typically contain, in addition to thepresent compounds, one or more of a sweetening agent, preservatives,dye/colorant and flavor enhancer. In a composition intended to beadministered by injection, one or more of a surfactant, preservative,wetting agent, dispersing agent, suspending agent, buffer, stabilizerand isotonic agent may be included.

Liquid pharmaceutical compositions of the disclosure, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. Parenteral preparations can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the disclosure intended foreither parenteral or oral administration should contain an amount of acompound of the disclosure such that a suitable dosage will be obtained.

Pharmaceutical compositions of the disclosure may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

Pharmaceutical compositions of the disclosure may be intended for rectaladministration, in the form, for example, of a suppository, which willmelt in the rectum and release the drug. Compositions for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

Pharmaceutical compositions of the disclosure may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

Pharmaceutical compositions of the disclosure may be prepared in dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of the disclosure may bedelivered in single phase, bi-phasic, or tri-phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols.

The pharmaceutical compositions of the disclosure may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining a compound of the disclosure with sterile,distilled water so as to form a solution. A surfactant may be added tofacilitate the formation of a homogeneous solution or suspension.Surfactants are compounds that non-covalently interact with the compoundof the disclosure so as to facilitate dissolution or homogeneoussuspension of the compound in the aqueous delivery system.

The compounds of the disclosure, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific compound employed; the metabolic stability and length ofaction of the compound; the age, body weight, general health, sex, anddiet of the patient; the mode and time of administration; the rate ofexcretion; the drug combination; the severity of the particular disorderor condition; and the subject undergoing therapy.

Compounds of the disclosure, or pharmaceutically acceptable derivativesthereof, may also be administered simultaneously with, prior to, orafter administration of one or more other therapeutic agents. Suchcombination therapy includes administration of a single pharmaceuticaldosage formulation which contains a compound of the disclosure and oneor more additional active agents, as well as administration of thecompound of the disclosure and each active agent in its own separatepharmaceutical dosage formulation. For example, a compound of thedisclosure and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof the disclosure and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in thesynthetic processes described herein the functional groups ofintermediate compounds may need to be protected by suitable protectinggroups. Such functional groups include hydroxy, amino, mercapto andcarboxylic acid. As described above, suitable protecting groups forhydroxy include trialkylsilyl or diarylalkylsilyl (for example,t-butyldimethylsilyl, 1-butyldiphenylsilyl or trimethylsilyl),tetrahydropyranyl, benzyl, and the like, and suitable protecting groupsfor amino, amidino and guanidino include t-butoxycarbonyl,benzyloxycarbonyl, and the like. Suitable protecting groups for mercaptoinclude —C(O)—R″ (where R″ is alkyl, aryl or arylalkyl),p-methoxybenzyl, trityl and the like. Suitable protecting groups forcarboxylic acid include alkyl, aryl or arylalkyl esters. Protectinggroups may be added or removed in accordance with standard techniques,which are known to one skilled in the art and as described herein. Theuse of protecting groups is described in detail in Green, T. W. and P.G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed.,Wiley. As one of skill in the art would appreciate, the protecting groupmay also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although aprotected derivative of compounds of this disclosure may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of thedisclosure which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisdisclosure are included within the scope of the present disclosure.

Furthermore, compounds of the disclosure which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of thedisclosure can be converted to their free base or acid form by standardtechniques.

The following Examples illustrate various methods of making compounds ofthis disclosure, i.e., compound of structures (I) and (II). It isunderstood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othercompounds of structure (I) and (II) not specifically illustrated belowby using the appropriate starting components and modifying theparameters of the synthesis as needed. In general, starting componentsmay be obtained from sources such as Sigma Aldrich, Lancaster Synthesis,Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedherein.

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES General Synthetic Schemes

General Procedure 1—Trifluoroacetamide Installation

To a stirred suspension of the amine in 1,4-dioxane was addedtrifluoroacetic anhydride (1.1 equivalents). The reaction mixturetransitioned from a suspension to a solution and back to a suspensionagain. The progress of the reaction was monitored by TLC and/or HPLC-MSfor completion. Once the starting material was fully consumed, thereaction was diluted with hexanes or diethyl ether, filtered on aBuchner funnel and the resulting solids were dried under reducedpressure to give the pure trifluoroacetamide.

General Procedure 2—DCC/DMAP Mediated N-Acyl Sulfonamide Formation

To a stirred solution of the acid in dichloromethane was added asolution of the sulfonamide (1.3 equivalents, in dichloromethane,N,N-dimethylformamide, or a mixture thereof, as necessary).Dicyclohexylcarbodiimide (1.2 equivalents) was added and subsequentlyN,N-dimethylaminopyridine (1.2 equivalents). Reaction course wasmonitored by HPLC-MS (typically 16 h) and excess by-products could beprecipitated by the addition of diethyl ether. Solids were removed byfiltration and washed with 1:1 diethyl ether/dichloromethane. Thecombined organic layers were concentrated, and the residue was purifiedby silica gel chromatography or optionally prep-HPLC to give the desiredN-acyl sulfonamide.

General Procedure 3—General saponification

To a solution of the trifluoroacetamide or ester containing construct in1,4-dioxane or methanol was added lithium hydroxide (10 equivalents) andwater (10% v/v). The reaction was allowed to stir at room temperature oroptionally heated to 50° C. Reaction course was monitored by HPLC-MS.Upon completion, volatiles were removed under reduced pressure, theaqueous layer was pH adjusted if necessary and washed successively withdichloromethane or ethyl acetate. The organic phases were pooled, driedover MgSO₄, filtered and concentrated. The reaction product was eitherused “as is” or purified by silica gel chromatography as necessary.

General Procedure 4—HATU Mediated Peptide Bond Formation

To a stirred solution of the carboxylic acid in a minimal amount ofdichloromethane or N,N-dimethylformamide or mixture thereof, at 0° C.was added HATU (equivalents) and N,N-diisopropylethylamine (4equivalents). Stirring was continued for a brief induction period (5-20minutes) at which time the reaction was charged with a solution of theamine in dichloromethane. The reaction was allowed to warm to roomtemperature and monitored for progress by HPLC-MS. Upon completion,volatiles were removed under reduced pressure and the residual materialwas purified by silica gel chromatography or reverse phase HPLC tofurnish amide in adequate purity.

General Procedure 7—Boc Group Removal

To a solution of the Boc-protected construct in dichloromethane wasadded 10% v/v trifluoroacetic acid. Reaction course was monitored byHPLC-MS. Upon completion, all volatiles were removed under reducedpressure. The residual material was purified either by reverse phaseHPLC, silica gel chromatography or precipitation from a mixture of coldmethanol/dichloromethane/diethyl ether.

General Procedure 8—Pd-Catalyzed Suzuki Cross Coupling

A suspension of aryl bromide, aryl (or alkenyl) boronic acid (1.5 eq),Pd(OAc)₂ (10 mol %), 2-(di-tert-butylphosphino)biphenyl (20 mol %), andK₃PO₄ (3 eq) in THF was stirred under N₂ at ambient temperature for 16 h(or 50° C. for 2 h). The resulting brown reaction mixture was dilutewith ether and washed with 1M NaOH (3×). The aqueous washes werecombined and extracted with ether (2×). The organics were combined,dried over MgSO₄, filtered, concentrated in vacuo and purified viasilica gel column chromatography (eluted with MeOH/CH₂Cl₂ mixtures) toafford the cross-coupled product.

General Procedure 9—Cu-Catalyzed Ullman Cross Coupling (MethoxyInstallation)

A mixture of aryl bromide, CuBr (20 mol %), NaOMe (20 eq, 4.9M in MeOH),and EtOAc (1.5 eq) was stirred under N₂ at 95° C. for 16 h. Theresulting mixture was diluted with H₂O and poured into cold (0° C.)stirring 1M citric acid. After stirring for 10 min, the mixture wasextracted with EtOAc (4×). The organics were combined, washed with H₂O(2×) and brine (1×), dried over MgSO₄, filtered and concentrated invacuo. The product was used in the next step without furtherpurification.

General Procedure 10—Vinylogous Amino Ester Synthesis

The procedure for Weinreb amide synthesis, reduction and subsequentolephination thereof as described by Nieman J. A. et al. J. Nat. Prod.2003, 66, 183-199 was employed to the desired commercially availableamino acids with no modifications.

General Procedure 11—Establishment of Boc-t-Leucine-(Me)-VinylogousAmino Acid

The vinylogous amino ester was deprotected and coupled to Boc-t-leucineaccording to procedures described by Nieman J. A. et al. J. Nat. Prod.2003, 66, 183-199 with no modifications.

General Procedure 12—Sulfonamide Formation from Alkyl Halide

To a suspension of the desired alkyl halide in 2:1 H₂O/EtOH was addedsodium sulfite (1.2 equiv). The resulting mixture was heated to refluxfor 6-24 h. The reaction was then cooled to room temperature, thesolvents were removed at reduced pressure to remove ethanol and theproduct was precipitated. The sodium alkylsulfonate were filtered,collected and dried in vacuo. These solids were then suspended indichloromethane and phosphorous pentachloride (2 equiv) was added withstirring. The resulting suspension was heated to reflux for 2 h andallowed to cool to room temperature. The reactions were then cooled to0° C. and water was added dropwise to consume excess phosphorouspentachloride. The mixture was transferred to a separatory funnel andthe organic phase was washed with brine, dried over MgSO₄, filtered andconcentrated to give the desired sulfonyl chloride. The thusly derivedchloride was subsequently dissolved in THF and added dropwise to astirred aqueous solution of concentrated ammonium hydroxide at 0° C.Upon completion of the addition, the reaction was concentrated underreduced pressure and diluted with water and ethyl acetate. The organicphase was washed with brine, dried over MgSO₄, filtered and concentratedto give the desired sulfonamide in sufficient purity for further use.

General Procedure 13—Sulfonamide Formation from Substituted ArylCompounds

To a stirred mixture of the desired aryl substituted compound inchloroform was added chlorosulfonic acid (4 equiv). The reaction washeated to 70° C. for 1 h and allowed to cool to room temperature.Thionyl chloride (2 equiv) was added and the reaction was again heatedto 70° C. for 1 h. The contents of the reaction vessel were concentratedunder reduced pressure to give an oil which was subsequently twicedissolved in toluene and concentrated under reduced pressure to removeresidual acid. The remaining material was dissolved in THF and addeddropwise to a concentrated, stirred solution of ammonium hydroxide at 0°C. Once the addition was complete, the reaction was concentrated underreduced pressure and the residue was partitioned between ethyl acetateand water. The organic phase was washed with brine, dried over MgSO₄,filtered and concentrated to give the desired phenylsulfonamide inadequate purity for further use.

General Procedure 14—Sulfonamide Formation

The procedures used to generate the desired sulfonamides were adaptedfrom Winum, J.-Y. et al., Org Lett, 2001, 3(14), 2241-2243

General Procedure 15—Preparation of MC-VC-PABC-Toxins

The appropriate intermediate amine or aniline was taken up in DMF (˜90mg/mL), and to this was added 1-hydroxybenzotriazole hydrate (0.3 eq),then commercially obtained MC-VC-PABC-PNP(4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl4-nitrophenyl carbonate) (1.3 eq) as described in Firestone, et al. U.S.Pat. No. 6,214,345 was added followed by pyridine (25 eq). The reactionwas covered to protect from light and stirred at ambient temperature for24 to 48 h. The reaction mixture could be purified by concentrating themixture and performing flash chromatography directly on the crude, oralternatively, it could be diluted with DMSO to an appropriate volumeand injected directly onto a preparatory HPLC to give the pure

MC-VC-PABC-R Construct.

All sulfonamides and sulfonamides or prescursors to the materials usedin the procedures below were purchased commercially and manipulated, ifnecessary, such that they were suitable for use. Specifically, GeneralProcedures 1, 12, 13 and 14 were employed to manipulate commerciallyavailable starting materials unless otherwise noted below. Sulfonamideanalogs of the N-acyl sulfonamide containing compounds disclosed hereinmay be synthesized by the artisan of reasonable skill based on theteachings herein and knowledge in the art, and are included within thescope of the invention.

Representative Compounds Example 1

To a stirred slurry of potassium bromide (1.904 g) in water (2.8 mL) wasadded 1,3-propanesultone. The reaction was heated to 60° C. withstirring for 1 h and allowed to cool to room temperature. Ethanol (˜45mL) was added with stirring and a precipitate formed. The suspension wasfiltered on a Buchner funnel and the solids were collected and dried athigh vacuum over night to give potassium 3-bromopropane-1-sulfonate(2.90 g, 12.0 mmol) as a white solid.

The above solid was added to a round bottom flask equipped with a stirbar. Phosphorous pentachloride (3.22 g, 1.3 equiv) was added in a singlecharge and the flask was gently shaken to mix the solids. A gas wasobserved to form and the solids became slightly molten. A singular dropof water was added to the mixture and a vigorous evolution of gas wasobserved, with more significant melting of the reaction mixture. Theflask was submerged in an oil bath at 70° C. and the molten mixturemanipulated to attempt to make it as uniform as possible. After 10minutes of heating, the flask was allowed to cool to room temperatureand was charged with ice (˜60 mL) and diethyl ether (˜80 mL) and stirredvigorously. The biphasic mixture was transferred to a separatory funnel,the organic layer washed with brine, then dried over MgSO₄, filtered andconcentrated to a total volume of ˜25 mL. The ethereal layer was addedto a 100 mL round bottom flask, a stir bar was added and the flask wascooled to 0° C. in an ice bath. Ammonia (NH₄OH, 28% aq, 5 mL) was addedwith vigorous stirring and an emulsion formed. After the emulsion hadsubsided, brine (˜20 mL) and diethyl ether (˜20 mL) were added and themixture transferred to a separatory funnel. The organic phase wasseparated, dried over MgSO₄ and concentrated to give the title compoundas a stiff syrup that solidified on standing (0.782 g).

¹H NMR (400 MHz, DMSO-d6) δ (ppm)=2.24 (p, 2H, J=6.5 Hz), 3.12 (t, 2H,J=6.5 Hz), 3.66 (t, 2H, J=6.5 Hz), 6.91 (s, 2H).

Example 2

To a stirred solution of triphenylmethanethiol (0.276 g) in N,N-dimethylformamide at 0° C. was added sodium hydride (0.04 g, 1 equiv). Aftereffervescence had ceased, 3-bromopropance-1-sulfonamide (0.100 g, 0.5equiv) was added as a solid in a single portion and the reaction wasallowed to warm to room temperature. Progress of the reaction wasmonitored by HPLC-MS and TLC (40% EtOAc in hexanes). After 2 h, thereaction was quenched with water (˜0.5 mL) and concentrated on a rotovapat high-vacuum. The resulting oil was partitioned between ethyl acetateand brine, transferred to a separatory funnel and the organic phase waswashed with brine, dried over MgSO₄, concentrated and purified by flashchromatography (5-50% EtOAc in hexanes) to give the title compound(0.135 g) as a white crystalline solid.

¹H NMR (400 MHz, CD3OD) δ (ppm)=1.77-1.85 (m, 2H), 2.35 (t, 2H, J=6.5Hz), 2.95-2.99 (t, 2H, J=6.5 Hz), 7.22-7.33 (m, 9H), 7.40-7.45 (m, 6H)

Example 3

(6S,9S,12S,E)-9-tert-butyl-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-phenylpropan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oicacid

Synthesized as per Nieman J. A. et al. J. Nat. Prod. 2003, 66, 183-199.

Example 4

(S,E)-N-(3-mercaptopropylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide(Compound A)

Example 4 was synthesized from Examples 2 and 3 according to GeneralProcedures 2 and 7 with the inclusion of tri-isoproypsilane (2equiv) toProcedure 9.

¹H NMR (400 MHz, CD3OD) δ (ppm)=0.88 (3H, d, J=6.2 Hz), 0.94 (3H, d,J=6.2 Hz), 1.08 (s, 9H), 1.40 (s, 3H), 1.48 (s, 3H), 1.94 (d, 3H, J=1.29Hz), 2.03-2.16 (m, 3H), 2.41 (s, 3H), 2.67 (t, 2H, J=9.76 Hz), 3.16 (s,3H), 3.46-3.50 (m, 2H), 4.08 (br s, 1H), 4.94 (s, 1H), 5.07 (t, 1H,J=10.0 Hz), 6.59 (d, 1H, J=9.5 Hz), 7.32-7.37 (m, 1H), 7.41-7.48 (m,2H), 7.50-7.57 (m, 2H).

Methods described above were used to generate the following analogouscompounds.

Example 5

Synthesized as described by Lemaire, H. and Rieger, M in J. Org. Chem.,1961, 1330-1331.

Example 6

(S,E)-N-(2-mercaptoethylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide(Compound B)

To a solution of(6S,9S,12S,E)-9-tert-butyl-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-phenylpropan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oicacid (0.138 g, 2.4 equiv) in dichloromethane (4 mL) was added2,2′-disulfanediyldiethanesulfonamide (0.028 g),di-isopropylcarbodiimide (0.044 mL, 2.4 equiv) and N,N-dimethylpyridine(0.034 g, 2.8 equiv). Stirring was continued for 16 h at which point TLCanalysis (5% MeOH (with 5% AcOH) in 70/30 CH₂Cl₂/Hexanes) indicatedcomplete consumption of the disulfanedisulfonamide. The reaction wasdiluted with hexanes (˜5 mL), filtered to remove solids, concentratedand the resultant oil purified by flash chromatography.

The chromatographically purified materials was then dissolved indichloromethane (3 mL), a stir bar was added, then trifluoroacetic acid(0.60 mL) and tri-isopropylsilane (0.20 mL). The mixture immediatelywent yellow, with the colour fading over 5 minutes and conversion of thematerial to the desired product was monitored by HPLC-MS. Upon completeconversion, the reaction was concentrated to dryness and the residuepurified by flash chromatography (0-15% MeOH (containing 5% AcOH) in80/20 CH₂Cl₂/hexanes). HPLC-MS showed this isolate to be a mixture offree thiol and disulfide.

1H NMR (400 MHz, CD3OD) δ (ppm)=0.88 (3H, d, J=6.2 Hz), 0.93 (3H, d,J=6.2 Hz), 1.07 (s, 9H), 1.40 (s, 3H), 1.47 (s, 3H), 1.91-2.05 (m, 5H),2.32 (s, 3H), 2.67 (t, 2H, J=9.76 Hz), 3.07-3.18 (m, 5H), 3.52-3.59 (m,2H), 3.85 (s, 1H), HH 4.08 (br s, 1H), 4.93 (s, 1H), 5.09 (t, 1H, J=10.0Hz), 6.76 (d, 1H, J=9.5 Hz), 7.29-7.35 (m, 1H), 7.39-7.46 (m, 2H),7.49-7.5 s (m, 2H). C₂₉H₄₈N₄O₅S₂ calcd. [M+H]⁺ =598.15 amu. foundm/z=598.16.

Example 7

To a stirred solution of triphenylmethanethiol (0.276 g, 2equiv) inN,N-dimethylformamide (3 mL) at 0° C. was added sodium hydride (60% w/wdispersion in mineral oil, 0.04 g, 2 equiv). When the effervescence hadceased, 4-(bromomethyl)benzenesulfonamide (0.125 g, 1 equiv) was addedin a single portion and the reaction was allowed to warm to roomtemperature. HPLC-MS at 20 minutes indicated that conversion wascomplete. The reaction was quenched with acetic acid (˜0.2 mL),concentrated to dryness in vacuo and the subsequent residue partitionedbetween ethyl acetate and brine. The organic layer was separated, driedover MgSO₄, filtered, concentrated and purified by flash chromatography(0-50% ethyl acetate in hexanes). Fractions containing the desiredmaterial were concentrated to dryness to furnish the desired compound asa colourless solid (0.200 g).

¹H NMR (400 MHz, DMSO-d6) δ (ppm)=3.38 (s, 2H), 7.24-7.35 (m, 7H),7.36-7.44 (m, 12H), 7.67-7.73 (m, 2H)

Example 8

(S,E)-N-(4-(mercaptomethyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide(Compound C)

Title compound prepared from Examples 3 and 7 according to GeneralProcedures 2 and 7

¹H NMR (400 MHz, CD3OD) δ (ppm)=0.88 (d, 3H, J=6.2 Hz), 0.91 (d, 3H,J=6.2 Hz), 1.06 (s, 9H), 1.38 (s, 3H), 1.47 (s, 3H), 1.86 (s, 3H),1.99-2.05 (m, 1H), 2.41 (s, 3H), 2.67 (t, 2H, J=9.76 Hz), 3.14 (s, 3H),3.80 (s, 2H), HH 4.10 (br s, 1H), 4.93 (s, 1H), 5.00 (t, 1H, J=10.0 Hz),6.54 (d, 1H, J=9.5 Hz), 7.30-7.51 (m, 5H), 7.52-7.58 (m, 2H), 7.90-7.97(m, 2H). C₃₄H₅₀N₄O₅S₂ calcd. [M+H]⁺=659.25 amu. found m/z=659.37.

Example 9

(S,E)-2,5-dimethyl-N-tosyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide(Compound D)

Title compound was prepared from Example 3 and tolylsulfonamide usingGeneral Procedures 2 and 7.

¹H NMR (400 MHz, CD3OD) δ (ppm)=0.88-0.94 (m, 6H), 1.06 (s, 9H), 1.35(s, 3H), 1.45 (s, 3H), 1.86 (s, 3H), 2.02-2.11 (m, 1H), 2.44 (s, 3H),2.51 (s, 3H), 3.17 (s, 3H), HH 4.35 (s, 1H), 4.89-4.99 (m, 2H), 6.48 (d,1H, J=9.5 Hz), 7.30-7.43 (m, 4H), 7.43-7.50 (m, 2H), 7.51-7.57 (m, 2H).C₃₄H₅₀N₄O₅S calcd. [M+H]⁺ =627.15 amu. found m/z=627.31.

Example 10

(S,E)-2,5-dimethyl-N-(methylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide(Compound E)

Title compound was prepared from Example 3 and methanesulfonamide usingGeneral Procedures 2 and 7.

¹H NMR (400 MHz, CD3OD) δ (ppm)=0.87-0.98 (3H (m, 6H), 1.09 (s, 9H),1.40 (s, 3H), 1.49 (s, 3H), 1.97 (s, 3H), 2.03-2.13 (m, 1H), 2.52 (s,3H), 2.67 (t, 2H, J=9.76 Hz), 3.18 (s, 3H), 3.31 (s, 3H), 4.38 (s, 1H),4.94 (d, 1H, J=8.2 Hz), 5.07 (t, 1H, J=10.0 Hz), 6.54 (d, 1H, J=9.5 Hz),7.30-7.40 (m, 1H), 7.40-7.51 (m, 2H), 7.51-7.59 (m, 2H). C₂₈H₄₆N₄O₅Scalcd. [M+H]⁺=551.30 amu. found m/z=551.34.

Example 11

(S,E)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoicacid (Compound F)

The title compound was synthesized using methods as described by Niemanet al. in J. Nat. Prod. 2003, 66, 183-199.

Example 12

(S,E)-N-(mesitylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and mesitylsulfonamide usingGeneral Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.60-7.55 (m, 2H), 7.47 (n, 2H), 7.37(m, 1H), 7.03 (s, 2H), 6.50 (d, J=6 Hz, 1H), 5.06-4.91 (m, 3H), 4.34 (s,1H), 3.17 (s, 3H), 2.68 (s, 6H), 2.51 (s, 3H), 2.31 (s, 3H), 2.07 (m,6.6 Hz, 2H), 1.87 (s, 3H), 1.48 (s, 3H), 1.36 (s, 3H), 1.09-1.04 (m,J=16.8 Hz, 10H), 0.92 (t, J=6.3 Hz, 6H).

C36H54N4O5S calcd m/z=654.38. found [M+H]+=655.03.

Example 13

(S,E)-2,5-dimethyl-N-(4-(trifluoromethoxy)phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and4-trifluoromethoxyphenylsulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.16 (dd, J=8.7, 1.4 Hz, 1H), 7.69-7.28(m, 4H), 6.52 (d, J=9.2 Hz, 1H), 5.02-4.95 (m, 1H), 4.92 (s, 0H), 4.35(s, 1H), 3.17 (s, 1H), 2.51 (s, 1H), 2.05 (ddd, J=15.9, 10.9, 3.7 Hz,1H), 1.87 (s, 1H), 1.47 (s, 1H), 1.36 (s, 1H), 1.07 (s, 4H), 0.91 (t,J=6.1 Hz, 3H).

C34H47F3N4O6S calcd m/z=696.32. found [M+H]+=697.26.

Example 14

(S,E)-N-(benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and benzylsulfonamide usingGeneral Procedures 2 and 7

1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J=7.9 Hz, 2H), 7.47 (t, J=7.3Hz, 2H), 7.38 (brs, 6H), 6.39 (d, J=9.4 Hz, 1H), 5.06 (t, J=10.0 Hz,1H), 4.93 (s, 1H), 4.75 (s, 2H), 4.36 (s, 1H), 3.13 (s, 3H), 2.51 (s,3H), 2.06-1.95 (m, 4H), 1.48 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.90(t, J=6.2 Hz, 6H).

C34H47F3N4O6S calcd m/z=626.35. found [M+H]+=626.99.

Example 15

(S,E)-2,5-dimethyl-N-(2,4,6-triisopropylphenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,4,6-tri-isopropylphenylsulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.61-7.53 (m, 2H), 7.47 (t, J=7.8 Hz,2H), 7.41-7.33 (m, 1H), 7.27 (s, 2H), 6.50 (dd, J=9.6, 1.8 Hz, 1H), 5.05(t, J=10.0 Hz, 1H), 4.92 (s, 1H), 4.43-4.26 (m, 3H), 3.16 (s, 3H), 2.94(dd, J=14.3, 7.4 Hz, 1H), 2.51 (s, 3H), 2.07-1.99 (m, 2H), 1.90 (d,J=1.4 Hz, 3H), 1.48 (s, 4H), 1.39 (s, 3H), 1.33-1.22 (m, 18H), 1.11 (s,2H), 1.06 (s, 9H), 0.91 (t, J=6.0 Hz, 7H).

C42H66N4O5S calcd m/z=738.48. found [M+H]+=738.10.

Example 16

(S,E)-N-(4-tert-butylphenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and4-tertbutylphenylsulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.98 (d, J=8.6 Hz, 2H), 7.64 (d, J=8.6Hz, 2H), 7.55 (d, J=7.9 Hz, 2H), 7.47 (t, J=7.7 Hz, 3H), 7.37 (t, J=7.1Hz, 1H), 6.48 (dd, J=9.6, 1.8 Hz, 1H), 4.99 (t, J=10.0 Hz, 1H), 4.92 (s,1H), 4.35 (s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 1.87 (d, J=1.4 Hz, 3H),1.47 (s, 3H), 1.38 (s, 10H), 1.06 (s, 9H), 0.91 (t, J=6.2 Hz, 7H).

C42H66N4O5S calcd m/z=668.40. found [M+H]+=669.28.

Example 17

(S,E)-N-(4-chlorophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 4-chlorophenylsulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.03 (d, J=8.7 Hz, 2H), 7.60 (d, J=8.7Hz, 2H), 7.57-7.51 (m, 2H), 7.47 (dd, J=8.6, 6.9 Hz, 2H), 7.42-7.32 (m,1H), 6.50 (dd, J=9.2, 1.7 Hz, 1H), 4.96 (dd, J=10.9, 9.1 Hz, 2H), 4.92(s, 1H), 4.35 (s, 1H), 3.17 (s, 3H), 2.51 (s, 3H), 2.14-2.03 (m, 1H),2.01 (s, 1H), 1.87 (d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.07(s, 9H), 0.91 (dd, J=6.5, 4.6 Hz, 7H).

C33H47C1N4O5S calcd m/z=646.30. found [M+H]+=647.20.

Example 18

(S,E)-N-(3-cyanophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 3-cyanophenylsulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.38 (s, 1H), 8.31 (dt, J=8.0, 1.5 Hz,1H), 8.02-7.92 (m, 1H), 7.75 (t, J=7.9 Hz, 1H), 7.53 (d, J=1.2 Hz, 1H),7.48 (dd, J=8.6, 6.9 Hz, 2H), 7.43-7.33 (m, 1H), 6.55 (dd, J=9.3, 1.7Hz, 1H), 4.93 (d, J=5.4 Hz, 2H), 4.35 (s, 1H), 3.18 (s, 3H), 2.51 (s,3H), 2.15-1.98 (m, 2H), 1.87 (d, J=1.4 Hz, 3H), 1.45 (s, 3H), 1.32 (s,3H), 1.07 (s, 9H), 0.92 (dd, J=6.6, 3.9 Hz, 7H).

C34H47N5O5S calcd m/z=637.33. found [M+H]+=638.00.

Example 19

(S,E)-2,5-dimethyl-N-(2-nitrophenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 2-nitrophenylsulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.36-8.27 (m, 1H), 7.82 (dd, J 5.9, 3.8Hz, 3H), 7.61-7.51 (m, 2H), 7.47 (dd, J=8.6, 6.9 Hz, 2H), 7.42-7.31 (m,1H), 6.63 (dd, J=9.5, 1.7 Hz, 1H), 5.03 (t, J=10.0 Hz, 1H), 4.93 (s,1H), 4.36 (s, 1H), 3.18 (s, 3H), 2.51 (s, 3H), 2.12-2.01 (m, 1H), 1.88(d, J=1.4 Hz, 3H), 1.48 (s, 3H), 1.37 (s, 3H), 1.06 (s, 9H), 0.97-0.86(m, 6H).

C34H47N5O5S calcd m/z=657.32. found [M+H]+=658.21.

Example 20

(S,E)-N-(4-methoxy-2-nitrophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound Was prepared from Example 3 and2-nitro-4-methoxyphenylsulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.24 (d, J=8.9 Hz, 1H), 7.59-7.51 (m,2H), 7.47 (t, J=7.6 Hz, 2H), 7.44-7.25 (m, 4H), 6.60 (dd, J=9.2, 1.7 Hz,1H), 5.03 (t, J=10.0 Hz, 1H), 4.93 (s, 1H), 4.36 (s, 1H), 3.97 (s, 3H),3.18 (s, 3H), 2.51 (s, 3H), 2.13-2.02 (m, 1H), 1.89 (d, J=1.4 Hz, 3H),1.48 (s, 3H), 1.38 (s, 3H), 1.11 (s, 2H), 1.06 (s, 9H), 0.99-0.88 (m,6H).

C34H49N5O8S calcd m/z=687.33. found [M+H]+=689.23.

Example 21

4-(N—((S,E)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)-3-nitrobenzamide

Title compound was prepared from Example 3 and3-nitro-4-sulfamoylbenzamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.35 (d, J=8.0 Hz, 1H), 8.22 (d, J=8.0Hz, 2H), 7.59-7.51 (m, 2H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.3 Hz,1H), 6.70-6.57 (m, 1H), 5.04 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.37 (s,1H), 3.17 (s, 3H), 2.52 (s, 3H), 2.05 (ddd, J=10.3, 7.4, 5.5 Hz, 1H),1.87 (d, J=1.4 Hz, 3H), 1.48 (s, 3H), 1.38 (s, 3H), 1.06 (s, 9H), 0.92(dd, J=14.7, 6.8 Hz, 6H).

C34H48N6O8S calcd m/z=700.33. found [M+H]+=701.28.

Example 22

(S,E)-N-(4-methoxyphenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and4-methoxyphenylsulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.97 (d, J=9.0 Hz, 2H), 7.54 (d, J=7.5Hz, 2H), 7.46 (t, J=7.6 Hz, 2H), 7.36 (t, J=7.2 Hz, 1H), 7.06 (d, J=9.0Hz, 2H), 6.48 (dd, J=9.3, 1.9 Hz, 1H), 4.97 (t, J=9.9 Hz, 1H), 4.92 (s,1H), 4.22 (s, 1H), 3.89 (s, 3H), 3.15 (s, 3H), 2.46 (s, 3H), 2.10-1.99(m, 2H), 1.86 (d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.06 (s,9H), 0.94-0.84 (m, 6H).

C34H50N4O6S calcd m/z=642.35. found [M+H]+=643.31.

Example 23

(S,E)-2,5-dimethyl-N-(4-(2,2,2-trifluoroacetamido)phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide using General Procedures2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.06 (d, J=8.9 Hz, 2H), 7.88 (d, J=8.9Hz, 2H), 7.52 (d, =7.1 Hz, 2H), 7.49-7.40 (m, 3H), 7.35 (dd, J=8.1, 6.1Hz, 1H), 6.47 (dd, J=9.2, 1.8 Hz, 1H), 4.33 (s, 1H), 3.15 (s, 3H), 2.48(s, 3H), 2.13-1.96 (m, 2H), 1.85 (d, J=1.4 Hz, 3H), 1.43 (s, 3H), 1.33(s, 3H), 1.04 (s, 9H), 0.89 (dd, J=6.8, 4.7 Hz, 6H).

C35H48F3N5O6S calcd m/z=723.33. found [M+H]+=724.08.

Example 24

(S,E)-N-(4-aminophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide using General Procedures2, 3 and 7

¹H NMR (400 MHz, Methanol-d₄) δ 7.71 (d, =8.8 Hz, 2H), 7.55 (d, J=7.6Hz, 2H), 7.47 (d, J=6.9 Hz, 2H), 7.37 (t, =6.8 Hz, 1H), 6.67 (d, J=8.8Hz, 2H), 6.44 (dd, J=9.2, 1.6 Hz, 1H), 4.97 (t, J=9.7 Hz, 1H), 4.92 (s,1H), 4.36 (s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 2.16-2.00 (m, 1H), 1.87(d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.92 (d,J=6.4 Hz, 3H), 0.91 (d, J=6.3 Hz, 3H).

C33H49N5O5S calcd m/z=627.35. found [M+H]+=628.35.

Example 25

(S,E)-2,5-dimethyl-N-(phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and phenylsulfonamide usingGeneral Procedures 2, and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.06-7.95 (m, 2H), 7.63-7.40 (m, 8H),7.40-7.30 (m, 1H), 6.53 (dd, J=9.3, 1.6 Hz, 1H), 5.05-4.95 (m, 1H), 4.22(s, 1H), 3.14 (s, 3H), 2.45 (s, 3H), 2.09-1.95 (m, 1H), 1.85 (d, J=1.4Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.06 (s, 9H), 0.89 (dd, J=11.9, 6.5Hz, 7H).

C33H48N4O5S calcd m/z=612.33. found [M+H]+=613.06.

Example 26

(S,E)-N—(N-(2-fluorobenzyl)sulfamoyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

2-fluorobenzylsulfamamide was prepared from 2-fluorobenzylamineaccording to General Procedure 14; the title compound was prepared fromExample 3 and 2-fluorobenzylsulfamamide using General Procedures 2 and7.

1H NMR (400 MHz, Methanol-d4) δ 7.63-7.41 (m, 6H), 7.41-7.26 (m, 3H),7.14 (td, J=7.5, 1.2 Hz, 1H), 7.07 (ddd, J=9.5, 8.2, 1.1 Hz, 1H), 6.37(dd, 9.4, 1.7 Hz, 1H), 5.07-4.97 (m, 1H), 4.37 (s, 1H), 4.33 (s, 2H),3.15 (s, 3H), 2.51 (s, 3H), 2.10-1.97 (m, 1H), 1.83 (d, J=1.4 Hz, 3H),1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.97-0.84 (m, 6H).

C34H50FN5O5S calcd m/z=659.35. found [M+H]+=660.28.

Example 27

(S,E)-2,5-dimethyl-N-(piperidin-1-ylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Piperidine-1-sulfonamide was synthesized from piperidine according toGeneral Procedure 14; the title compound was prepared from Example 3 andpiperidine-1-sulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.55 (d, J=1.2 Hz, 1H), 7.47 (t, J=7.6Hz, 3H), 7.42-7.29 (m, 1H), 6.48 (dd, J=9.7, 1.8 Hz, 1H), 5.05 (t,J=10.0 Hz, 1H), 4.39 (s, 1H), 3.18 (s, 3H), 2.52 (s, 3H), 2.07 (d,J=10.5 Hz, 1H), 1.96 (d, J=1.4 Hz, 3H), 1.61 (ddd, J=20.0, 10.3, 5.4 Hz,9H), 1.49 (s, 4H), 1.39 (s, 3H), 1.09 (s, 9H), 0.99-0.84 (m, 9H).

C32H53N5O5S calcd m/z=619.38. found [M+H]+=620.38.

Example 28

(S,E)-2,5-dimethyl-N-(o-tolylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 2-toluenesulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.10 (dd, J=8.0, 1.4 Hz, 1H), 7.60-7.33(m, 11H), 6.52 (dd, J=9.6, 1.7 Hz, 1H), 5.04-4.90 (m, 2H), 4.35 (s, 1H),3.18 (s, 3H), 2.67 (s, 3H), 2.51 (s, 3H), 2.15-2.03 (m, 2H), 2.01 (s,1H), 1.87 (d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.35 (s, 3H), 1.07 (s, 9H),0.92 (t, J=6.3 Hz, 6H).

C34H50N4O5S calcd m/z=626.35. found [M+H]+=627.05.

Example 29

(S,E)-N-(4-bromophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 4-bromophenylsulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.95 (d, J=8.3 Hz, 2H), 7.76 (d, J=8.0Hz, 2H), 7.55 (d, J=7.5 Hz, 2H), 7.47 (dd, J=8.6, 6.9 Hz, 2H), 7.41-7.29(m, 1H), 6.51 (d, J=9.0 Hz, 1H), 4.35 (s, 1H), 3.16 (s, 3H), 2.50 (s,3H), 2.06 (dt, J=10.7, 6.3 Hz, 1H), 1.87 (s, 3H), 1.46 (s, 3H), 1.36 (s,3H), 1.07 (s, 9H), 0.91 (dd, J=6.9, 4.9 Hz, 8H).

C33H47BrN4O5S calcd m/z=690.25. found [M+H]+=691.17, 693.18.

Example 30

(S,E)-2,5-dimethyl-N-(naphthalen-2-ylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 2-naphthylsulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.69-8.62 (m, 1H), 8.47 (d, J=8.2 Hz,1H), 8.14-7.95 (m, 5H), 7.71 (dddd, J=18.4, 8.2, 6.9, 1.4 Hz, 2H),7.57-7.50 (m, 2H), 7.46 (dd, J=8.6, 6.9 Hz, 2H), 7.42-7.33 (m, 1H), 6.50(dd, J=9.3, 1.5 Hz, 1H), 4.92-4.87 (m, 1H), 4.34 (s, 1H), 3.16 (s, 3H),2.50 (s, 3H), 2.13-1.99 (m, 1H), 1.85 (d, J=1.4 Hz, 3H), 1.44 (s, 3H),1.34 (s, 3H), 1.04 (s, 9H), 0.90 (dd, J=6.6, 4.0 Hz, 6H).

C37H50N4O5S calcd m/z=662.35. found [M+H]+=663.32.

Example 31

methyl4-(N—((S,E)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzoate

Title compound was prepared from Example 3 and4-carboxymethylphenylsulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.24-8.10 (m, 4H), 7.58-7.50 (m, 2H),7.47 (dd, J=8.6, 6.9 Hz, 2H), 7.41-7.33 (m, 1H), 6.52 (dd, J=9.2, 1.6Hz, 1H), 4.35 (s, 1H), 3.97 (s, 3H), 3.18 (s, 3H), 2.50 (s, 31-0,2.15-2.00 (m, 1H), 1.86 (d, J=1.4 Hz, 3H), 1.45 (s, 3H), 1.35 (s, 3H),1.07 (s, 9H), 0.91 (dd, J=6.7, 3.8 Hz, 6H).

C35H50N4O7S calcd m/z=670.34. found [M+H]+=671.10.

Example 32

(S,E)-2,5-dimethyl-N—(N-(2-(trifluoromethyl)benzyl)sulfamoyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2-trifluoromethylbenzylsulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.78 (d, J=7.9 Hz, 1H), 7.74-7.67 (m,1H), 7.64 (dd, J=8.1, 6.7 Hz, 1H), 7.60-7.52 (m, 2H), 7.48 (dd, J=8.5,6.8 Hz, 4H), 7.42-7.33 (m, 1H), 6.48-6.40 (m, 1H), 5.11-5.02 (m, 1H),4.45 (s, 2H), 4.37 (s, 1H), 3.17 (s, 3H), 2.52 (s, 3H), 2.11-1.99 (m,2H), 1.92 (d, J=1.4 Hz, 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.09 (s, 9H),0.92 (dd, J=9.3, 6.7 Hz, 6H).

C35H50F3N5O5S calcd m/z=709.35. found [M+H]+=710.02.

Example 33

(4S,E)-N-(hexan-2-ylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and hexane-2-sulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.56-7.48 (m, 2H), 7.42 (t, J=7.8 Hz,2H), 7.31 (t, J=7.3 Hz, 1H), 6.58-6.50 (m, 1H), 5.05 (t, J=10.0 Hz, 1H),4.92 (s, 1H), 3.84 (s, 1H), 3.65 (dt, J=10.8, 4.3 Hz, 1H), 3.14 (s, 3H),2.32 (s, 3H), 2.09-1.96 (m, 2H), 1.93 (d, J=1.4 Hz, 3H), 1.61-1.27 (m,3H), 1.06 (s, 9H), 0.98-0.90 (m, 6H), 0.87 (d, J=6.5 Hz, 3H).

C33H56N4O5S calcd m/z=620.40. found [M+H]+=621.55.

Example 34

(S,E)-N-(2-methoxyethylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2-methoxyethanesulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J=7.8 Hz, 2H), 7.47 (t, =7.6Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 6.51 (d, J=9.4 Hz, 1H), 5.07 (t, J=10.0Hz, 1H), 4.95 (s, 1H), 4.33 (s, 1H), 3.82 (t, J=5.8 Hz, 2H), 3.70 (q,J=5.2 Hz, 2H), 3.18 (s, 3H), 2.50 (s, 3H), 2.18-2.00 (m, 1H), 1.95 (d,J=1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.93 (dd,J=14.8, 6.6 Hz, 6H).

C30H50N4O6S calcd m/z=594.35. found [M+H]+=595.44.

Example 35

(S,E)-N-(cyclopentylmethylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 andcyclopentylmethanesulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.61-7.52 (m, 2H), 7.48 (dd, J=8.6, 6.9Hz, 2H), 7.38 (t, J=7.4 Hz, 1H), 6.54 (dd, J=9.4, 1.7 Hz, 1H), 5.06 (t,J=10.0 Hz, 1H), 4.94 (s, 1H), 4.37 (s, 1H), 3.52 (dd, J=7.0, 5.4 Hz,3H), 3.18 (s, 3H), 2.52 (s, 3H), 2.35 (p, J=8.1 Hz, 1H), 2.16-1.89 (m,6H), 1.77-1.53 (m, 4H), 1.49 (s, 3H), 1.45-1.26 (m, 5H), 1.09 (s, 9H),0.93 (dd, J=11.3, 6.7 Hz, 6H).

C33H54N4O5S calcd m/z=618.38. found [M+H]+=619.54.

Example 36

(S)-methyl2-(tert-butoxycarbonyl(methyl)amino)-3-(4-cyanophenyl)-3-methylbutanoate

To a mixture of the methyl ester of Example 38 (0.06 g, 0.15 mmol),tris(dibenzylideneacetone)dipalladium(0) (0.014 g, 0.015 mmol),1,1′-Bis(diphenylphosphino)ferrocene (0.02 g, 0.25 equiv), magnesiumacetate (0.013 g, 0.06 mmol), zinc dust (0.004 g, 0.06 mmol) and zinccyanide (0.0264 g, 0.225 mmol) under a bath of nitrogen was addedN,N-dimethylformamide/water (0.8/0.08 mL). The reaction was sparged withnitrogen gas, then the vial was sealed and immersed in an oil bath at105° C. The reaction was allowed to stir overnight and allowed to coolto room temperature. HPLC-MS analysis indicated good conversion to thedesired product. The reaction was concentrated at reduced pressure,suspended in CH₂Cl₂ and the resulting suspension purified by silica gelchromatography (15-25% EtOAc in Hexanes) to yield the final compound asa colourless oil (0.036 g, 69%).

1H NMR (400 MHz, Chloroform-d) δ 7.69-7.35 (m, 4H), 5.24 (s, 1H), 3.54(s, 3H), 2.74 (s, 3H), 1.51 (s, 3H), 1.45-1.25 (m, 12H).

Example 37

(S)-methyl2-(tert-butoxycarbonyl(methyl)amino)-3-(4-((tert-butoxycarbonylamino)methyl)phenyl)-3-methylbutanoate

To a solution of the benzonitrile (0.300 g, 0.87 mmol) inmethanol/acetic acid (10:1, 9 mL) in a shaker vessel was added palladiumblack. The flask was charged with hydrogen gas at 60 psi and the shakerturned on for 24 h. At that time, the vessel was purged of H₃ underreduced pressure. The reaction was diluted with methanol and thesuspension filtered through a celite pad. The filtrate was concentratedto a slightly yellow oil and re-dissolved in dichloromethane (5 mL).t-butyl dicarbonate (0.524 g, 2.0 equiv) and triethylamine (0.846 mL, 5equiv) were added to the solution at 0° C. with stirring. The reactionwas allowed to stir for 3 h at which time HPLC-MS indicated completeconsumption of the amine. The reaction was concentrated under reducedpressure and purified by silica gel chromatography (diethyl ether inhexanes, 15-30%) to yield the title compound as a colourless oil (0.232g, 60%).

1H NMR (400 MHz, Chloroform-d) δ 7.38 (dd, J=16.6, 8.0 Hz, 2H), 7.23 (d,J=7.7 Hz, 2H), 5.27 (s, 1H), 4.31 (s, 2H), 3.61 (s, 3H), 2.78 (s, 3H),1.50-1.61 (m, 6H), 1.47 (d, J=15.2 Hz, 18H).

Example 38

(S)-3-(4-bromophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoicacid

To a stirred solution of (S)-methyl3-(4-bromophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoate(0.710 g, 1.77 mmol) in 1,4 dioxane (4 mL) was added water (1 mL) (2 mL)and lithium hydroxide monohydrate (0.367 g, 8.9 mmol). The reaction washeated to 50° C. and monitored by HPLC for completion. The reaction wascooled to room temperature, acidified to pH 3 with 1M citric acid andconcentrated to near dryness under reduced pressure. The residue wastaken up in ˜20 mL ethyl acetate, washed with brine, dried over MgSO₄,filtered and concentrated to give analytically pure material that wasused without further manipulation.

1H NMR (400 MHz, Chloroform-d) δ 7.44 (d, J=8.3 Hz, 2H), 7.33 (d, J=8.3Hz, 2H), 5.18 (s, 1H), 2.71 (s, 3H), 1.60-1.42 (m, 15H).

Example 39

(S)-3-(4-azidophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoicacid

To an open pressure tube containing a magnetic stir bar was addedExample 38 (0.690 g, 1.8 mmol), copper(I) iodide (0.034 g, 0.18 mmol),sodium azide (0.350 g, 5.4 mmol), N1,N2-dimethylethane-1,2-diamine(0.029 mL, 0.27 mmol), sodium ascorbate (0.036 g, 0.18 mmol), sodiumhydroxide (0.072 g, 1.8 mmol), ethanol (6 mL) and water (1 mL). Thesuspension was sparged with nitrogen gas, the vessel was sealed andimmersed in an oil bath at 105° C. with vigorous stirring. The course ofreaction was monitored by HPLC-MS over the course of 24 h at which timelittle starting material remained. The reaction was diluted with ethylacetate (˜20 mL) and washed with brined. The aqueous layer was extracted2× with ˜20 mL ethyl acetate. The organic layers were combined, driedover MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography (20-65% EtOAc(containing 2% v/v AcOH) in hexanes) to give the title compound as acolourless oil (0.475 g, 75%).

1H NMR (400 MHz, Chloroform-d) δ 7.44 (d, J=8.6 Hz, 2H), 6.99 (dd,J=9.0, 3.4 Hz, 2H), 5.24 (s, 1H), 2.71 (s, 3H), 1.63-1.38 (m, 18H).

Example 40

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(4-cyanophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared from Example 36 and(S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamideusing General Procedures 3, 4 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.83 (d, J=8.2 Hz, 2H), 7.73 (d, J=8.4Hz, 2H), 7.38 (d, J=2.6 Hz, 5H), 6.39 (dd, J=9.2, 1.8 Hz, 1H), 5.04 (t,J=10.1 Hz, 1H), 4.91 (s, 1H), 4.75 (s, 2H), 4.34 (s, 1H), 3.12 (s, 3H),2.54 (s, 3H), 2.05-1.97 (m, 2H), 1.95 (d, J=1.5 Hz, 3H), 1.52 (s, 3H),1.41 (s, 3H), 1.09 (s, 9H), 0.91 (dd, J=11.2, 4.8 Hz, 6H).

C₃₅H₄₉N₅O₅S calcd m/z=651.35. found [M+H]⁺ =652.4.

Example 41

(S,E)-4-((S)-2-((S)-3-(4-(aminomethyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide

Title compound was prepared from Example 37 and(S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamideusing General Procedures 3, 4 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.63 (t, J=8.8 Hz, 2H), 7.54 (d, J=8.3Hz, 2H), 7.49-7.43 (m, 3H), 7.39 (m, 2H), 6.39 (d, J=9.4 Hz, 1H),5.05-4.97 (m, 1H), 4.75 (s, 2H), 4.35 (s, 3H), 4.16 (s, 2H), 3.14 (s,3H), 2.54 (s, 3H), 2.03 (m, 1H), 1.95 (s, 3H), 1.51 (s, 3H), 1.39 (s,3H), 1.31 (s, 3H), 1.09 (s, 9H), 0.98-0.81 (m, 6H).

Example 42

(S,E)-4-((S)-2-((S)-3-(4-azidophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide

Title compound was prepared from Example 39 and(S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamideusing General Procedures 4 and 7.

C₃₄H₄₉N₇O₅S calcd m/z=667.35 amu. found [M+H]⁺=668.4.

Example 43

(S,E)-4-((S)-2-((S)-3-(4-aminophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide

To a stirred solution of Boc protected Example 42 (0.035 g, 0.046 mmol)in ethanol (1.6 mL) and water (0.5 mL) was added zinc dust (0.015 g,0.23 mmol) and ammonium chloride (0.025 g, 0.46 mmol). After 1 h HPLC-MSindicated complete consumption of the starting material. The reactionwas quenched with ammonium hydroxide (˜0.1 mL) and diluted with ethylactetate (5 mL). The reaction was filtered, the solids washed with ethylacetate (5 mL) and the biphasic filtrate transferred to a separatoryfunnel. The aqueous phase was washed twice with ethyl acetate (5 mL) andthe organic phases were combined, washed with brine, dried over MgSO₄,filtered and concentrated. The reaction product was purified by silicagel chromatography (5-15% MeOH in CH₂Cl₂) to afford the Boc protectedintermediate as a colouless glass (0.027 g, 66%). The intermediate wasdeprotected according to General Procedure 7 to give the title compound.

C₃₄H₅₁N₅O₅S calcd m/z=641.36 amu. found [M+H]⁺ =642.4.

Example 44

(S,E)-N-(cyclohexylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and cyclohexylsulfonamideusing General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.61-7.52 (m, 2H), 7.47 (dd, J=8.6, 6.9Hz, 2H), 7.36 (t, J=7.5 Hz, 1H), 6.61-6.50 (m, 1H), 5.11-4.99 (m, 1H),4.94 (s, 1H), 4.28 (s, 1H), 3.59-3.51 (m, 1H), 3.18 (s, 3H), 2.48 (s,3H), 2.20-2.00 (m, 4H), 1.97-1.87 (m, 6H), 1.78-1.69 (m, 1H), 1.60 (td,J=14.2, 10.9 Hz, 2H), 1.48 (s, 3H), 1.44-1.23 (m, 6H), 1.09 (s, 9H),0.93 (dd, J=13.7, 6.6 Hz, 7H).

C33H54N4O5S calcd m/z=618.38. found [M+H]+=619.47.

Example 45

(S,E)-2,5-dimethyl-N-(pyridin-3-ylmethylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 andpyridin-3-ylmethanesulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.55 (d, J=1.7 Hz, 1H), 8.48 (dd, J=5.0,1.6 Hz, 1H), 7.89 (d, J=8.0 Hz, 0H), 7.55 (d, J=7.6 Hz, 2H), 7.50-7.39(m, 2H), 7.35 (s, 1H), 6.52 (dd, J=9.6, 2.0 Hz, 1H), 5.05 (s, 0H), 4.94(s, 1H), 4.64 (s, 2H), 4.19 (s, 1H), 3.11 (s, 3H), 2.45 (s, 3H), 1.91(d, J=1.5 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.07 (s, 8H), 0.89 (dd,J=15.1, 6.5 Hz, 6H).

C33H54N4O5S calcd m/z=627.35. found [M+H]+=628.35.

Example 46

4-(N—((S,E)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzoicacid

Title compound was prepared from Example 3 and methyl4-sulfamoylbenzoate using General Procedures 2, 3 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.25-8.07 (m, 4H), 7.54 (d, J=7.8 Hz,2H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 6.55 (d, J=9.3 Hz,1H), 4.98 (t, J=9.9 Hz, 1H), 4.92 (s, 1H), 4.36 (s, 1H), 3.16 (s, 3H),2.51 (s, 3H), 2.06 (q, J=9.0, 7.7 Hz, 1H), 1.88 (s, 3H), 1.46 (s, 3H),1.36 (s, 3H), 1.06 (s, 9H), 0.91 (t, J=6.0 Hz, 6H)

Example 47

(S,E)-2,5-dimethyl-N-(3-(2,2,2-trifluoroacetamido)phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(3-sulfamoylphenyl)acetamide using General Procedures2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.49 (p, J=2.2 Hz, 1H), 7.90 (dtd, =6.0,4.8, 2.9 Hz, 2H), 7.64-7.56 (m, 1H), 7.53 (tt, J=5.4, 4.3, 1.8 Hz, 2H),7.51-7.42 (m, 2H), 7.41-7.28 (m, 1H), 6.56-6.38 (m, 1H), 4.97 (s, 1H),4.90 (d, J=3.3 Hz, 1H), 4.35 (s, 1H), 3.16 (d, J=15.5 Hz, 3H), 2.49 (d,J=14.2 Hz, 3H), 2.14-2.01 (m, 1H), 1.89-1.83 (m, 3H), 1.57-1.28 (m, 6H),1.14-0.94 (m, 9H), 0.95-0.85 (m, 6H).

¹³C NMR (101 MHz, Methanol-d₄) δ 172.26, 168.81, 167.10, 167.00, 144.95,141.82, 138.82, 138.47, 135.31, 130.71, 130.38, 128.91, 127.36, 126.65,126.32, 121.39, 71.20, 66.92, 57.87, 57.78, 42.05, 35.83, 34.15, 32.66,30.84, 29.79, 26.95, 21.39, 19.84, 19.82, 15.45, 14.03.

¹⁹F NMR (377 MHz, Methanol-d₄) δ −76.96, −77.07.

C₃₅H₄₈F₃N₅O₆S calcd m/z=723.33 amu. found [M+H]⁺ =724.30,[M+Na]⁺=746.30.

Example 48

(S,E)-N-(3-aminophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(3-sulfamoylphenyl)acetamide using General Procedures2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.55 (d, J=7.5 Hz, 2H), 7.51-7.45 (m,2H), 7.43-7.20 (m, 4H), 6.97 (d, J=8.1 Hz, 1H), 6.48 (d, J=9.4 Hz, 1H),5.02-4.89 (m, 2H), 4.36 (s, 1H), 3.17 (s, 3H), 2.50 (s, 3H), 2.14-2.00(m, 1H), 1.88 (d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.35 (s, 3H), 1.07 (s,9H), 0.92 (d, J=6.3 Hz, 3H), 0.90 (s, 3H).

C₃₃H₄₉N₅O₅S calcd. m/z=627.35. found [M+H]⁺=628.36.

Example 49

(S,E)-2,5-dimethyl-N-(pyridin-3-ylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and pyridine-3-sulfonamideusing General Procedures 2, and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 9.18 (s, 1H), 8.80 (s, 1H), 8.46 (dt,J=8.2, 1.8 Hz, 1H), 7.65 (dd, J=8.1, 4.9 Hz, 1H), 7.54 (d, J=7.3 Hz,2H), 7.47 (t, J=7.8 Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 6.54 (d, J=9.3 Hz,1H), 5.01-4.88 (m, 2H), 4.36 (s, 1H), 3.18 (s, 3H), 2.51 (s, 3H),2.15-2.01 (m, 1H), 1.86 (d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.33 (s, 3H),1.07 (s, 9H), 0.92 (d, J=3.3 Hz, 3H), 0.91 (d, J=3.5 Hz, 3H).

C₃₂H₄₇N₅O₅S calcd. m/z=613.33. found [M+H]⁺=614.23.

Example 50

(S,E)-2,5-dimethyl-N-(thiophen-2-ylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and thiophene-2-sulfonamideusing General Procedures 2, and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.93-7.82 (m, 2H), 7.55 (d, J=8.3 Hz,1H), 7.48 (t, J=7.8 Hz, 2H), 7.37 (t, J=7.2 Hz, 1H), 7.15 (dd, J=5.0,3.8 Hz, 1H), 6.51 (d, J=9.1 Hz, 1H), 5.02-4.93 (m, 2H), 4.36 (s, 1H),3.18 (s, 3H), 2.51 (s, 3H), 2.15-2.01 (m, 1H), 1.89 (d, J=1.4 Hz, 3H),1.46 (s, 3H), 1.34 (s, 3H), 1.08 (s, 9H), 0.93 (d, J=4.8 Hz, 3H), 0.91(d, J=4.7 Hz, 3H).

C₃₁H₄₆N₄O₅S₂ calcd. m/z=618.29. found [M+H]⁺ =619.24.

Example 51

(S,E)-N-(4-hydroxyphenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and4-(tert-butyldimethylsilyloxy)benzenesulfonamide using GeneralProcedures 2, and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.89 (d, J=8.8 Hz, 2H), 7.55 (d, J=7.0Hz, 1H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 6.91 (d, J=8.9Hz, 2H), 6.46 (d, J=9.2 Hz, 1H), 4.97 (d, J=10.2 Hz, 1H), 4.92 (s, 1H),4.33 (s, 1H), 3.16 (s, 3H), 2.50 (s, 3H), 2.11-2.00 (m, 1H), 1.87 (d,J=1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.07 (s, 9H), 0.92 (d, J=6.5Hz, 4H), 0.89 (d, J=6.7 Hz, 3H).

C₃₃H₄₈N₄O₆S calcd. m/z=628.33. found [M+H]⁺=629.38.

Example 52

Tritylmercaptan (1.48 g, 5.36 mmol, 1.05 eq) in THF (5 mL) was addeddropwise to a stirred suspension of sodium hydride (60% dispersion inmineral oil, 214 mg, 5.36 mmol, 1.05 eq) in THF (5 mL) under N₂ at 0° C.After 15 min, 4-(bromomethyl)benzonitrile (1.00 g, 5.10 mmol, 1.0 eq) inTHF (5 mL) was added and the reaction was allowed to come to rt. After 1h, TLC indicated complete conversion of starting material. The reactionwas quenched by adding saturated ammonium chloride, then some dH₂O. Themixture was extracted three times with ether, washed with saturatedbrine, dried over sodium sulfate, and concentrated to a viscous yellowoil. Purification by flash chromatography gave the title compound (1.76g, 88%) as a light white powder.

¹H NMR (400 MHz, Chloroform-d) δ 7.52 (d, J=8.2 Hz, 2H), 7.47 (d, =7.1Hz, 6H), 7.33 (t, =7.5 Hz, 6H), 7.26 (t, J=7.2 Hz, 3H), 7.19 (d, =8.2Hz, 2H), 3.40 (s, 2H). m/z calcd. for C₂₇H₂₁NS=391.14. Found[M+Na]⁺=414.13. R_(f)=0.32 (10% EtOAc/Hex).

Example 53

4-(tritylthiomethyl)benzonitrile (1.47 g, 3.75 mmol, 1.0 eq) was takenup in 40 mL THF, under N₂ atmosphere, then cooled to −78° C. To thissolution was added Ti(O-iPr)₄ (1.21 mL, 4.13 mmol, 1.1 eq), thenethylmagnesium bromide (3 M, 2.75 mL, 8.26 mmol, 2.2 eq) was addeddropwise over 5 min. The dry-ice bath was removed, allowing the solutionto reach rt. After 45 min at rt, BF₃.Et₂O (0.93 mL, 7.51 mmol, 2.0 eq)was added to the now very dark reaction mixture. After stirring for anadditional 2.5 h, the reaction was quenched with 5 mL of 2 M HCl,followed by pH adjustment to strong base with about 15 mL 2 M NaOH. Somewater was added to the mixture, then it was extracted three times with75 mL EtOAc, washed once with dH₂O, once with saturated brine, driedover sodium sulfate, and concentrated to a clear oil. The material waspurified by flash chromatography to afford the title compound (680 mg,36%) as a clear oil.

¹H NMR (400 MHz, Chloroform-d) δ 7.49 (d, J=7.8 Hz, 6H), 7.33 (t, J=7.7Hz, 6H), 7.26 (t, J=7.2 Hz, 3H), 7.20 (d, J=8.2 Hz, 2H), 7.11 (d, J=8.2Hz, 2H), 3.32 (s, 2H), 1.06 (dd, J=7.9, 5.0 Hz, 2H), 0.95 (dd, J=7.9,4.7 Hz, 2H). m/z calcd. for C₂₉H₂₇NS=421.19. Found [M+H]⁺=422.19.R_(f)=0.21 (50% EtOAc/Hex).

Example 54

To a stirred solution of 1-(4-(tritylthiomethyl)phenyl)cyclopropanamine(680 mg, 1.61 mmol, 1.0 eq) in CH₂Cl₂ was added trifluoroaceticanhydride (0.448 mL, 3.22 mmol, 2.0 eq) and triethylamine (0.45 mL, 3.22mmol, 2.0 eq). After two hours, TLC and HPLC indicated completeconversion of starting material. The reaction was quenched by theaddition of 3 mL NaHCO₃, then some dH₂O was added, and the mixture wasextracted three times with CH₂Cl₂. The combined organics were washedwith saturated brine, dried over sodium sulfate, and concentrated to ayellow foam, giving the title compound (715 mg, 86%) in sufficientpurity to move to the next step.

1H NMR (400 MHz, Chloroform-d) δ 7.48 (d, J=7.7 Hz, 6H), 7.32 (t, =7.6Hz, 6H), 7.25 (t, J=7.2 Hz, 3H), 7.19 (d, J=8.2 Hz, 2H), 7.10 (d, J=8.3Hz, 2H), 6.83 (s, 1H), 3.31 (s, 2H), 1.40-1.24 (m, 4H). m/z calcd. forC₃₁H₂₆F₃NOS=517.17. Found [M+Na]⁺=540.25. R_(f)=0.71 (50% EtOAc/Hex).

Example 55

2,2,2-trifluoro-N-(1-(4-(tritylthiomethyl)phenyl)cyclopropyl)acetamide(715 mg, 1.38 mmol, 1.0 eq) in 5 mL CH₂Cl₂ was treated with 2.5 mL TFA.After 1 min, TIPSH (0.42 mL, 2.1 mmol, 1.5 eq) was added, causing theyellow color to fade. After 30 min, TLC indicated the reaction to becomplete. The mixture was concentrated, then co-evaporated once withCH₂Cl₂ and twice with toluene. The residue was purified by flashchromatography to afford the title compound (261 mg, 69%) as a whitesolid. ¹H NMR (400 MHz, Chloroform-d) δ 7.35-7.23 (m, 4H), 6.87 (s, 1H),3.74 (d, J=7.6 Hz, 2H), 1.77 (t, J=7.6 Hz, 1H), 1.36 (s, 4H). R_(f)=0.47(20% EtOAc/Hex).

Example 56

2,2,2-trifluoro-N-(1-(4-(sulfamoylmethyl)phenyl)cyclopropyl)acetamide

To a stirred solution of2,2,2-trifluoro-N-(1-(4-(mercaptomethyl)phenyl)cyclopropyl)acetamide(220 mg, 0.799 mmol, 1.0 eq) in acetonitrile were added dH₂O (0.029 mL,1.6 mmol, 2.0 eq), tetrabutylammonium chloride (110 mg, 0.40 mmol, 0.5eq), then N-chlorosuccinimide (320 mg, 2.40 mmol, 3.0 eq). After 20minutes, no starting material was visible by TLC. After 90 min,concentrated NH₄OH (0.18 mL, 3.2 mmol, 4.0 eq) was added. After 10minutes, 1 mL of NH₄Cl was added, and the mixture was extracted threetimes with EtOAc. The combined organics were washed twice with dH₂O,once with saturated brine, dried over sodium sulfate, and concentratedto a clear oil. The residue was purified by flash chromatography toafford the title compound (192 mg, 74%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.21 (s, 1H), 7.31 (d, J=8.2 Hz, 2H), 7.16(d, J=8.3 Hz, 2H), 6.85 (s, 2H), 4.23 (s, 2H), 1.27 (dt, J=6.1, 2.3 Hz,4H). R_(f)=0.26 (50% EtOAc/Hex).

Example 57

(S,E)-2,5-dimethyl-N-(4-(1-(2,2,2-trifluoroacetamido)cyclopropyl)benzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and Example 56 using GeneralProcedures 2, and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.56 (d, J=8.4 Hz, 2H), 7.48 (t, J=7.7Hz, 2H), 7.37 (t, J=7.4 Hz, 1H), 7.32 (d, J=8.5 Hz, 2H), 7.28 (d, J=8.5Hz, 2H), 6.37 (d, J=9.6 Hz, 1H), 5.07 (t, J=10.0 Hz, 1H), 4.94 (s, 1H),4.72 (s, 2H), 4.37 (s, 1H), 3.13 (s, 3H), 2.52 (s, 3H), 2.08-1.96 (m,1H), 1.96 (d, 0.1=1.5 Hz, 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.35-1.27 (m,4H), 1.10 (s, 9H), 0.92 (d, J=7.1 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H).

¹³C NMR (101 MHz, MeOD) δ 170.93, 168.81, 165.64, 143.58, 142.24,136.87, 134.19, 130.64, 129.00, 127.63, 127.53, 125.95, 125.61, 69.90,57.10, 57.02, 56.39, 40.73, 34.55, 34.25, 32.80, 30.60, 29.33, 28.39,25.57, 20.11, 18.38, 18.34, 16.21, 16.15, 14.04, 12.85.

C₃H₅₄F₃N₅O₆S calcd. m/z=777.37. found [M+H]⁺=778.55.

Example 58

(S,E)-N-(4-(1-aminocyclopropyl)benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and Example 56 using GeneralProcedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.56 (d, J=8.7 Hz, 2H), 7.51 (s, 4H),7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 6.49 (d, J=9.5 Hz, 1H),5.07 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.81 (d, J=14.0 Hz, 1H), 4.77 (d,J=13.8 Hz, 1H), 4.39 (s, 1H), 3.16 (s, 3H), 2.52 (s, 3H), 2.11-1.99 (n,1H), 1.97 (d, 0.1=1.5 Hz, 3H), 1.49 (s, 8H), 1.45-1.41 (m, 2H), 1.40 (s,3H), 1.34-1.26 (m, 2H), 1.10 (s, 9H), 0.93 (d, J=6.2 Hz, 3H), 0.90 (d,J=6.3 Hz, 3H).

¹³C NMR (101 MHz, MeOD) δ 170.94, 169.00, 165.69, 143.57, 137.54,137.12, 134.38, 131.43, 129.66, 128.98, 127.51, 125.98, 69.85, 65.51,57.68, 57.15, 56.39, 40.72, 36.16, 34.51, 32.80, 30.68, 29.42, 28.40,25.61, 20.14, 18.42, 18.39, 14.05, 12.86, 11.80.

C₃₇H₅₅N₅O₅S calcd. m/z=681.39. found [M+H]⁺=682.49.

Example 59

The title compound was prepared as described in Beaus, P., Szymoniak, J.J. Org. Chem., 2003, 68, 7133-7136 from benzonitrile (1.0 mL, 9.7 mmol)to give 270 mg (21%).

¹H NMR (400 MHz, Chloroform-d) δ 7.44-7.28 (m, 4H), 7.27-7.15 (m, 1H),1.18-1.06 (m, 2H), 1.07-0.95 (m, 2H). R_(f)=0.28 (5% (5%NH₄OH/MeOH)/CH₂Cl₂).

Example 60

To a stirred solution of 1-phenylcyclopropanamine (270 mg, 2.03 mmol,1.0 eq) in dioxane (5 mL), was added trifluoroacetic anhydride (0.310mL, 2.23 mmol, 1.1 eq). After 5 min, TLC indicated complete conversionof starting material. The mixture was concentrated, then coevaporatedonce with CH₂Cl₂ and once with toluene to yield the title compound (453mg, 97%) as a flaky white powder.

¹H NMR (400 MHz, Chloroform-d) δ 7.47-7.15 (m, 5H), 6.88 (s, 1H), 1.65(s, 4H). m/z calcd. for C₁₁H₁₀F₃N0=229.07. Found [M+H]⁺ =230.14.R_(f)=0.82 (5% (5% NH₄OH/MeOH)/CH₂Cl₂).

Example 61

To stirred chlorosulfonic acid (0.78 mL, 11.8 mmol, 6.0 eq) at 0° C.,was added solid 2,2,2-trifluoro-N-(1-phenylcyclopropyl)acetamide (450mg, 1.96 mmol, 1.0 eq) portionwise, keeping the temperature low. Aftercomplete addition, the mixture was heated to 50° C. After 10 minutes,gas evolution ceased, and the reaction was allowed to cool. The mixturewas added slowly to a beaker of ice, being mindful of splattering. Thesolid that was left in the ice was filtered off. This solid was dried invacuo and then taken up in THF (4 mL). Concentrated NH₄OH (0.44 mL, 7.85mmol, 4.0 eq) was added, turning the solution green-black. After 2 min,TLC indicated complete consumption of the sulfonylchloride intermediate.2M HCl was added until the color faded, then the mixture was extractedthree times with EtOAc, washed once with saturated NaHCO₃, once withsaturated brine, dried over sodium sulfate, and concentrated to a flakysolid. The crude material was purified by flash chromatography to yieldthe title compound (235 mg, 39%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 7.76 (d, J=8.5 Hz, 2H), 7.32(d, J=8.1 Hz, 2H), 7.31 (s, 2H), 1.42-1.35 (m, 2H), 1.35-1.27 (m, 2H).m/z calcd. for C₁₁H₁₁F₃N₂O₃S=308.04. Found [M+H]⁺=309.07. R_(f)=0.27(50% EtOAc/Hex).

Example 62

(S,E)-2,5-dimethyl-N-(4-(1-(2,2,2-trifluoroacetamido)cyclopropyl)phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and Example 61 using GeneralProcedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.00 (d, J=8.6 Hz, 2H), 7.55 (d, J=7.6Hz, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.48-7.33 (m, 4H), 6.47 (dd, J=9.4, 1.6Hz, 1H), 5.00 (t, J=10.0 Hz, 1H), 4.92 (s, 1H), 4.35 (s, 1H), 3.15 (s,3H), 2.51 (s, 3H), 2.11-2.00 (m, 1H), 1.86 (d, J=1.4 Hz, 3H), 1.47 (d,J=6.2 Hz, 3H), 1.45 (s, 2H), 1.43 (s, 2H), 1.38 (s, 3H), 1.06 (s, 9H),0.91 (d, J=6.1 Hz, 3H), 0.89 (d, J=6.2 Hz, 3H).

C₃₇H₅₀F₃N₅O₆S calcd. m/z=763.36. found [M+H]⁺=764.45.

Example 63

(S,E)-N-(4-(1-aminocyclopropyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido) hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(1-(4-sulfamoylphenyl)cyclopropyl)acetamide usingGeneral Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.13 (d, J=8.5 Hz, 2H), 7.66 (d, J=8.6Hz, 2H), 7.55 (d, =7.2 Hz, 2H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.2Hz, 1H), 6.50 (dd, J=9.4, 1.7 Hz, 1H), 5.02 (t, J=10.0 Hz, 1H), 4.93 (d,J=4.9 Hz, 1H), 4.38 (s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 2.12-1.99 (m,1H), 1.84 (d, J=1.4 Hz, 3H), 1.51-1.46 (m, 5H), 1.46-1.42 (On, 2H), 1.38(s, 3H), 1.07 (s, 9H), 0.91 (dd, J=6.7, 1.7 Hz, 6H).

C₃₆H₅₃N₅O₅S calcd. m/z=667.38. found [M+H]⁺=668.40.

Example 64

(S,E)-2,5-dimethyl-N-(2-methylbenzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 2-methylbenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.61-7.52 (m, 2H), 7.48 (t, J=7.6 Hz,2H), 7.37 (t, J=7.3 Hz, 1H), 7.30-7.23 (m, 3H), 7.22-7.14 (m, 1H), 6.48(dd, =9.3, 1.7 Hz, 1H), 5.08 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.81 (s,2H), 4.34 (s, 1H), 3.15 (s, 3H), 2.51 (s, 3H), 2.48 (s, 3H), 2.08-2.00(m, 1H), 1.98 (d, J=1.1 Hz, 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.10 (s,9H), 0.93 (d, J=6.6 Hz, 3H), 0.91 (d, J=6.6 Hz, 3H).

C₃₅H₅₂N₄O₅S calcd. m/z=640.37. found [M+H]⁺=641.41.

Example 65

(S,E)-2,5-dimethyl-N-(4-nitrobenzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 4-nitrobenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.18 (d, J=8.7 Hz, 2H), 7.64 (d, =8.7Hz, 2H), 7.52 (d, J=7.5 Hz, 2H), 7.42 (t, J=7.7 Hz, 2H), 7.31 (t, J=7.3Hz, 1H), 6.55 (d, J=9.4 Hz, 1H), 5.04 (t, J=10.0 Hz, 1H), 4.92 (s, 1H),4.63 (s, 2H), 3.08 (s, 3H), 2.32 (s, 3H), 1.95 (dt, J=11.4, 6.6 Hz, 4H),1.89 (d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.38 (s, 3H), 1.05 (s, 9H), 0.89(d, J=6.5 Hz, 3H), 0.85 (d, J=6.5 Hz, 3H).

C₃₄H₄₉N₅O₇S calcd. m/z=671.34. found [M+H]⁺ =672.36.

Example 66

(S,E)-N-(4-chlorobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 4-chlorobenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.56 (d, J=7.9 Hz, 2H), 7.48 (t, J 7.6Hz, 2H), 7.44-7.34 (m, 5H), 6.39 (d, J=9.5 Hz, 1H), 5.06 (t, J=10.0 Hz,1H), 4.94 (s, 1H), 4.75 (s, 2H), 4.35 (s, 1H), 3.13 (s, 3H), 2.51 (s,3H), 2.06-1.95 (m, 1H), 1.95 (d, J=1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s,3H), 1.09 (s, 9H), 0.91 (d, J=6.1 Hz, 3H), 0.89 (d, J=5.9 Hz, 3H).

C₃₄H₄₉ClN₄O₅S calcd. m/z=660.31. found [M+H]⁺ =661.32.

Example 67

(S,E)-2,5-dimethyl-N-(phenethylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and homobenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.56 (d, J=7.6 Hz, 2H), 7.48 (t, J=7.5Hz, 2H), 7.38 (t, J=7.4 Hz, 1H), 7.34-7.28 (m, 2H), 7.28-7.20 (m, 3H),6.47 (dd, J=9.2, 1.7 Hz, 1H), 5.03 (t, J=10.0 Hz, 1H), 4.94 (s, 1H),4.36 (d, J=2.3 Hz, 2H), 3.78 (td, J=7.5, 4.1 Hz, 2H), 3.17 (s, 3H), 3.12(t, J=7.8 Hz, 2H), 2.51 (s, 3H), 2.14-2.01 (m, 1H), 1.89 (d, J=1.4 Hz,3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.94 (d, J=6.6 Hz, 3H),0.91 (d, J=6.6 Hz, 3H).

C₃₅H₅₂N₄O₅S calcd. m/z=640.37. found [M+H]⁺ =641.36.

Example 68

(S,E)-N-(4-bromobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 4-bromobenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.60-7.51 (m, 4H), 7.48 (t, J=7.7 Hz,2H), 7.39 (s, 1H), 7.31 (d, J=8.3 Hz, 2H), 6.38 (d, J=9.3 Hz, 1H), 5.06(t, J=10.0 Hz, 1H), 4.93 (s, 1H), 4.74 (s, 2H), 4.36 (s, 1H), 3.13 (s,3H), 2.52 (s, 3H), 2.03-1.98 (m, 1H), 1.95 (d, J=1.4 Hz, 3H), 1.49 (s,3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.91 (d, J=6.1 Hz, 3H), 0.89 (d, J=6.3Hz, 3H)

C₃₄H₄₉BrN₄O₅S calcd. m/z=704.26. found [M+H]⁺ =705.23.

Example 69

(S,E)-N-(4-cyanobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 4-cyanobenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.77 (d, J=8.3 Hz, 2H), 7.64-7.53 (m,4H), 7.48 (t, 7.7 Hz, 2H), 7.38 (t, J=7.3 Hz, 1H), 6.41 (dd, =9.3, 1.7Hz, 1H), 5.05 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.87 (s, 2H), 4.36 (s,1H), 3.14 (s, 3H), 2.52 (s, 3H), 2.06-1.98 (m, 1H), 1.95 (d, =1.4 Hz,3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.91 (d, J=4.0 Hz, 3H),0.90 (d, J=4.0 Hz, 3H).

C₃₅H₄₉N₅O₅S calcd. m/z=651.35. found [M+H]⁺=652.38.

Example 70

(S,E)-2,5-dimethyl-N-(3-nitrobenzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 3-nitrobenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.29 (d, J=8.0 Hz, 1H), 8.26 (s, 1H),7.83 (d, J=7.8 Hz, 1H), 7.67 (t, J=8.0 Hz, 1H), 7.56 (d, J=7.2 Hz, 2H),7.48 (t, J=7.7 Hz, 2H), 7.38 (t, J=7.3 Hz, 1H), 6.43 (dd, J=9.4, 1.7 Hz,1H), 5.05 (t, J=10.0 Hz, 1H), 4.93 (s, 2H), 4.93 (s, 1H), 4.36 (s, 1H),3.13 (s, 3H), 2.52 (s, 3H), 2.08-1.98 (m, 1H), 1.96 (d, J=1.4 Hz, 3H),1.48 (s, 3H), 1.39 (s, 3H), 1.07 (s, 9H), 0.89 (d, J=6.6 Hz, 3H), 0.88(d, J=6.6 Hz, 3H).

C₃₄H₄₉N₅O₇S calcd. m/z=671.34. found [M+H]⁺ =672.39.

Example 71

(S,E)-N-(4-tert-butylbenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and4-t-butylbenzylsulfonamide using General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.56 (d, J=7.6 Hz, 2H), 7.48 (t, J=7.7Hz, 2H), 7.43 (d, J=8.2 Hz, 2H), 7.38 (t, J=7.3 Hz, 1H), 7.30 (d, J=8.2Hz, 2H), 6.39 (dd, J=9.4, 1.6 Hz, 1H), 5.07 (t, J=10.0 Hz, 1H), 4.93 (s,1H), 4.72 (s, 2H), 4.37 (s, 1H), 3.13 (s, 3H), 2.52 (s, 3H), 2.06-1.98(m, 1H), 1.96 (d, J=1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.33 (s,9H), 1.10 (s, 9H), 0.92 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.5 Hz, 3H).

C₃₈H₅₈N₄O₅S calcd. m/z=682.41. found [M+H]⁺=683.47.

Example 72

(S,E)-2,5-dimethyl-N-(2-nitrobenzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 2-nitrobenzylsulfonamideusing General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.03 (dd, J=8.0, 1.4 Hz, 1H), 7.72 (td,J=7.5, 1.5 Hz, 1H), 7.65 (td, J=7.7, 1.6 Hz, 1H), 7.60 (dd, J=7.6, 1.6Hz, 1H), 7.56 (d; J=7.2 Hz, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.38 (t, J=7.3Hz, 1H), 6.43 (dd, J=9.4, 1.6 Hz, 1H), 5.31 (d, J=14.2 Hz, 1H), 5.26 (d,J=15.3 Hz, 1H), 5.06 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.37 (s, 1H),3.15 (s, 3H), 2.52 (s, 3H), 2.08-1.98 (m, 1H), 1.96 (d, J=1.4 Hz, 3H),1.49 (s, 3H), 1.39 (s, 3H), 1.10 (s, 9H), 0.92 (d, J=6.6 Hz, 3H), 0.90(d, J=6.6 Hz, 3H).

C₃₄H₄₉N₅O₇S calcd. m/z=671.34. found [M+H]=672.39.

Example 73

(S,E)-2,5-dimethyl-N-(4-nitrophenethylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and4-nitro-homobenzyisulfonamide using General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 8.19 (d, J=8.7 Hz, 2H), 7.58-7.51 (m,4H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 6.47 (dd, J=9.5,1.7 Hz, 1H), 5.00 (t, J=10.0 Hz, 1H), 4.93 (s, 1H), 4.36 (s, 1H), 3.91(dd, J=14.9, 8.5 Hz, 1H), 3.84 (dd, J=12.9, 8.5 Hz, 1H), 3.28 (t, J=7.5Hz, 2H), 3.16 (s, 3H), 2.51 (s, 3H), 2.12-1.98 (m, 1H), 1.87 (d, J=1.4Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.08 (s, 9H), 0.91 (d, J=6.6 Hz,3H), 0.91 (d, J=6.6 Hz, 3H).

C35H51N507S calcd. m/z=685.35. found [M+H]+=686.38.

Example 74

methyl4-chloro-3-(N—((S,E)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzoate

Title compound was prepared from Example 3 and methyl4-chloro-3-sulfamoylbenzoate using General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.80 (d, J=2.1 Hz, 1H), 8.20 (dd, J=8.3,2.1 Hz, 1H), 7.71 (d, J=8.3 Hz, 1H), 7.59-7.52 (m, 2H), 7.47 (t, J=7.7Hz, 2H), 7.40-7.32 (m, 1H), 6.63-6.56 (m, 1H), 5.02 (t, J=10.0 Hz, 1H),4.37 (s, 1H), 3.98 (s, 3H), 3.18 (s, 3H), 2.51 (s, 3H), 2.13-2.00 (m,1H), 1.86 (d, J=1.4 Hz, 3H), 1.47 (s, 3H), 1.37 (s, 3H), 1.06 (s, 9H),0.96-0.87 (m, 6H).

¹³C NMR (101 MHz, Methanol-d₄) δ 170.87, 165.65, 164.87, 143.61, 137.01,136.04, 134.29, 133.23, 131.81, 129.16, 128.98, 128.88, 127.50, 125.98,69.81, 65.53, 57.39, 56.35, 56.15, 55.37, 51.86, 40.70, 34.51, 32.77,30.80, 29.39, 28.44, 26.18, 25.56, 20.06, 18.40, 14.06, 12.74.

C₃₅H₄₉C1N₄O₇S calcd m/z=704.30 amu. found [M+H]⁺ =705.25,[M+Na]⁺=727.25.

Example 75

2,2,2-trifluoro-N-(4-(sulfamoylmethyl)benzyl)acetamide

The title compound was synthesized from commercially available(4-(aminomethyl)phenyl)methanesulfonamide and TFAA using GeneralProcedure 1.

¹H NMR (400 MHz, Acetone-d₆) δ 9.05 (s, 1H), 7.48-7.40 (m, 2H),7.40-7.32 (m, 2H), 6.17 (s, 1H), 4.56 (d, J=6.1 Hz, 2H), 4.35 (s, 2H)

Example 76

(S,E)-2,5-dimethyl-N-(4-((2,2,2-trifluoroacetamido)methyl)benzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and Example 75 using GeneralProcedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.57-7.49 (m, 2H), 7.45 (t, J=7.5 Hz,2H), 7.33 (p, J=8.8, 7.9 Hz, 5H), 6.37 (d, J=9.7 Hz, 1H), 5.09-5.00 (m,1H), 4.69 (s, 2H), 4.44 (s, 2H), 4.30 (s, 1H), 3.10 (s, 3H), 2.45 (d,J=17.5 Hz, 3H), 2.02-1.87 (m, 4H), 1.46 (s, 3H), 1.37 (s, 3H), 1.07 (s,9H), 0.95-0.81 (m, 6H).

¹⁹F NMR (377 MHz, Methanol-d₄) δ −76.94, −77.24.

C₃₇H₅₂F₃N₅O₆S calcd m/z=751.36 amu. found [M+H]⁺=752.46, [M+Na]⁺=774.38.

Example 77

(S,E)-N-(4-(aminomethyl)benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Prepared from Example 3 and Example 75 using General Procedures 2, 3 and7

¹H NMR (400 MHz, Methanol-d₄) δ 7.60-7.54 (n, 2H), 7.54-7.50 (m, 4H),7.47 (d, J=8.1 Hz, 2H), 7.37 (t, J=7.4 Hz, 1H), 6.49 (dd, J=9.5, 1.5 Hz,1H), 5.07 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.83 (d, J=14.3 Hz, 1H),4.79 (d, J=13.9 Hz, 1H), 4.38 (s, 1H), 4.16 (s, 2H), 3.16 (s, 3H), 2.52(s, 3H), 2.10-2.00 (m, 1H), 1.97 (d, J=1.4 Hz, 3H), 1.49 (s, 3H), 1.40(s, 3H), 1.10 (s, 9H), 0.93 (d, J=6.9 Hz, 3H), 0.91 (d, J=7.0 Hz, 3H).

C₃₅H₅₃N₅O₅S calcd. m/z=655.4. found [M+H]⁺=656.3, [M+2H]²⁺=328.8.

Example 78

The title compound was synthesized from commercially available(4-aminophenyl)methanesulfonamide and TFAA using General Procedure 1.

¹H NMR (400 MHz, DMSO-d₆) δ 11.31 (s, 1H), 7.79-7.51 (n, 2H), 7.51-7.23(n, 2H), 6.85 (s, 2H), 4.27 (s, 2H).

Example 79

(S,E)-2,5-dimethyl-N-(4-(2,2,2-trifluoroacetamido)benzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and Example 78 using GeneralProcedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.68 (d, J=8.6 Hz, 2H), 7.54 (d, J=7.1Hz, 2H), 7.45 (t, J=7.6 Hz, 2H), 7.37 (dd, J=10.6, 5.0 Hz, 3H), 6.34 (d,J=9.4 Hz, 1H), 5.04 (t, J=10.1 Hz, 2H), 4.74 (s, 2H), 4.35 (s, 1H), 3.10(s, 3H), 2.49 (s, 3H), 2.02-1.94 (m, 1H), 1.93 (d, J=1.4 Hz, 3H), 1.46(s, 3H), 1.37 (s, 3H), 1.06 (s, 9H), 0.88 (d, J=6.3 Hz, 3H), 0.86 (s,3H).

¹⁹F NMR (377 MHz, Methanol-d₄) δ −76.97, −77.05.

C₃₆H₅₀F₃N₅O₆S calcd m/z=737.34 amu. found [M+H]⁺ =738.38,[M+Na]⁺=760.35.

Example 80

(S,E)-N-(4-aminobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and Example 78 using GeneralProcedures 2, 3 and 7

¹H NMR (400 MHz, Methanol-d₄) δ 7.56 (d, J=7.6 Hz, 2H), 7.48 (t, J=7.7Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 7.20 (d, J=8.5 Hz, 2H), 6.87 (d, J=8.5Hz, 2H), 6.39 (d, J=9.4 Hz, 1H), 5.07 (t, J=10.0 Hz, 1H), 4.95 (s, 1H),4.64 (s, 2H), 4.38 (s, 1H), 3.14 (s, 3H), 2.52 (s, 3H), 2.07-1.98 (m,1H), 1.96 (d, J=1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.10 (s, 9H),0.92 (d, J=6.7 Hz, 3H), 0.90 (d, J=6.4 Hz, 3H).

C₃₄H₅₁N₅O₅S calcd. m/z=641.4. found [M+H]⁺=642.3.

Example 81

To a stirred solution of 4-(bromomethyl)benzenesulfonamide (0.50 g) inN,N-dimethylformamide (1 mL) was added sodium azide (0.20 g). Thesuspension was heated to 50° C. for 3 hours at which points the solventwas removed under reduced pressure. The residue was partitioned betweenethyl acetate and water. The organic phase was washed with brine, driedover magnesium sulfate, filtered and concentrated to dryness to give thetitle compound as a syrup that solidified on standing.

¹H NMR (400 MHz, Chloroform-d) δ 8.06-7.91 (m, 2H), 7.58-7.44 (m, 2H),4.96 (s, 2H), 4.48 (s, 2H).

Example 82

To a solution of 4-(azidomethyl)benzenesulfonamide (0.354 g) in methanol(10 mL) in a round bottom flask equipped with a magnetic stirrer wasadded 10% Pd/C (˜0.05 g). The flask was evacuated of gases at reducedpressure and charged with hydrogen. This evacuation and charge wasrepeated three times at which point the suspension was left to stirovernight. At 16 h, TLC analysis indicated complete consumption of thestarting material. The reaction was diluted with methanol (40 mL),celite was added and the mixture was filtered through a fritted glassfunnel. The resulting solution was concentrated to dryness. ¹H NMRsuggested that the material was sufficiently clean at this stage forfurther use without purification.

¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (m, 2H), 7.53 (m, 2H), 5.76 (s, 2H),3.76 (d, =11.9 Hz, 2H).

Example 83

The title compound was synthesized by reaction of4-(aminomethyl)benzenesulfonamide with TFAA according to GeneralProcedure 1, with a ¹H NMR spectrum that was complicated by rotamers.

¹H NMR (400 MHz, DMSO-d₆) δ 7.91-7.75 (m, 2H), 7.55-7.31 (m, 4H), 4.72(m, 2H), 4.47 (d, J=6.0 Hz, 1H), 3.18 (s, 2H).

Example 84

(S,E)-2,5-dimethyl-N-(4-((2,2,2-trifluoroacetamido)methyl)phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamdo)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and Example 83 using GeneralProcedures 2 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.02 (d, J=8.5 Hz, 2H), 7.58-7.42 (m,7H), 7.35 (t, J=7.3 Hz, 1H), 6.46 (d, J=8.5 Hz, 1H), 4.97 (d, J=10.4 Hz,1H), 4.54 (s, 2H), 4.33 (s, 1H), 3.14 (s, 3H), 2.48 (s, 3H), 2.11-1.97(m, 1H), 1.83 (d, J=1.4 Hz, 3H), 1.53 (s, 1H), 1.44 (s, 3H), 1.34 (s,3H), 1.04 (s, 9H), 0.89 (d, J=3.9 Hz, 3H), 0.88 (d, J=4.1 Hz, 3H).

¹⁹F NMR (377 MHz, Methanol-d₄) δ −76.94, −77.26.

C₃₆H₅₀F₃N₅O₆S calcd m/z=737.34 amu. found [M+H]⁺=738.39, [M+Na]⁺=760.41.

Example 85

(S,E)-N-(4-(aminomethyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Prepared from Example 3 and Example 83 using General Procedures 2, 3 and7

¹H NMR (400 MHz, Methanol-d₄) δ 8.13 (d, J=8.3 Hz, 2H), 7.68 (d, J=8.3Hz, 2H), 7.55 (d, J=7.6 Hz, 2H), 7.47 (t, J=7.7 Hz, 2H), 7.37 (t, J=7.3Hz, 1H), 6.51 (dd, J=9.2, 1.8 Hz, 1H), 5.01 (t, J=10.0 Hz, 1H), 4.37 (s,1H), 4.24 (s, 2H), 3.17 (s, 3H), 2.51 (s, 3H), 2.13-1.97 (m, 1H), 1.84(d, J=1.4 Hz, 3H), 1.47 (s, 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.91 (dd,J=6.7, 2.0 Hz, 7H).

C₃₄H₅₁N₅OS calcd m/z=641.36 amu. found [M+H]⁺=642.4.

Example 86

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(4-bromophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared from Example 38 and(S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamideusing General Procedures 4 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.62 (t, J=9.2 Hz, 2H), 7.50-7.43 (m,2H), 7.38 (d, J=2.2 Hz, 5H), 6.38 (dd, J=9.5, 1.8 Hz, 1H), 5.05 (t,J=10.0 Hz, 1H), 4.92 (s, 1H), 4.75 (d, J=2.2 Hz, 2H), 4.30 (s, 1H), 3.12(s, 3H), 2.53 (s, 3H), 2.06-1.97 (m, 1H), 1.95 (d, J=1.5 Hz, 3H), 1.47(s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.94-0.86 (m, 6H).

C₃₄H₄₉BrN₄O₅S calcd m/z=704.26 amu. found [M+H]⁺=705.29, [M+Na]=727.36.

Example 87

(S,E)-4-((S)-2((S)-3-(4′-acetylbiphenyl-4-yl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide

Title compound was prepared according to General Procedure 8 from Bocprotected Example 86 and 4-acetylphenylboronic acid.

¹H NMR (400 MHz, Methanol-d₄) δ 8.15-8.08 (m, 2H), 7.86-7.76 (m, 4H),7.66 (dd, J=14.7, 8.4 Hz, 2H), 7.38 (d, J=4.9 Hz, 5H), 6.39 (d, J=9.3Hz, 1H), 5.05 (t, J=10.1 Hz, 1H), 4.94 (s, 1H), 4.75 (d, J=4.1 Hz, 2H),4.37 (d, J=16.1 Hz, 1H), 3.13 (d, J=3.4 Hz, 3H), 2.67 (s, 3H), 2.53 (d,J=11.6 Hz, 3H), 2.01 (s, 1H), 1.96 (d, J=1.5 Hz, 3H), 1.54 (d, J=3.7 Hz,3H), 1.44 (s, 3H), 1.09 (d, J=2.7 Hz, 9H), 0.96-0.83 (m, 6H).

C₄₂H₅₆N₄O₆S calcd m/z=744.39 amu. found [M+H]⁺=745.42, [M+Na]⁺=767.36.

Example 88

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(4′-methoxybiphenyl-4-yl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared according to General Procedure 8 from Bocprotected Example 86 and 4-methoxyphenylboronic acid.

¹H NMR (400 MHz, Methanol-d₄) δ 7.74-7.53 (m, 6H), 7.38 (d, J=4.7 Hz,5H), 7.08-6.99 (m, 2H), 6.43-6.35 (m, 1H), 5.06 (s, 1H), 4.94 (s, 1H),4.75 (d, J=4.1 Hz, 2H), 4.38 (s, 1H), 3.86 (s, 3H), 3.13 (s, 3H), 2.54(s, 3H), 1.99 (d, J=11.0 Hz, 1H), 1.96 (d, J=1.5 Hz, 3H), 1.51 (s, 3H),1.43 (s, 3H), 1.09 (s, 9H), 0.96-0.85 (m, J=6.0, 5.1 Hz, 6H).

C₄₁H₅₆N₄O₆S calcd m/z=732.39 amu. found [M+H]⁺=733.41, [M+Na]⁺=755.40.

Example 89

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(biphenyl-4-yl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared according to General Procedure 8 from Bocprotected Example 86 and phenylboronic acid.

¹H NMR (400 MHz, Methanol-d₄) δ 7.86-7.51 (m, 6H), 7.48 (t, J=7.6 Hz,2H), 7.43-7.33 (m, 6H), 6.39 (d, J=9.5 Hz, 1H), 5.06 (t, J=10.1 Hz, 1H),4.94 (s, 1H), 4.75 (d, J=3.3 Hz, 2H), 4.37 (d, J=14.4 Hz, 1H), 3.13 (d,J=3.7 Hz, 3H), 2.55 (d, J=4.5 Hz, 3H), 2.06-1.97 (m, 1H), 1.96 (d, =1.5Hz, 3H), 1.52 (s, 3H), 1.44 (d, J=4.5 Hz, 3H), 1.09 (d, J=5.6 Hz, 9H),0.96-0.83 (m, 6H).

C₄₀H₅₄N₄O₅S calcd m/z=702.38 amu. found [M+H]⁺ =703.40, [M+Na]⁺=725.45.

Example 90

(S,E)-N-(benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-(4-(4-methylstyryl)phenyl)butanamido)butanamido)hex-2-enamide

Title compound was prepared according to General Procedure 8 from Bocprotected Example 86 and (E)-4-methylstyrylboronic acid.

¹H NMR (400 MHz, Methanol-d₄) δ 7.65 (d, J=8.2 Hz, 2H), 7.54 (d, J=8.2Hz, 2H), 7.47 (d, J=7.8 Hz, 2H), 7.38 (s, 5H), 7.26-7.11 (m, 4H), 6.39(d, J=9.3 Hz, 1H), 5.06 (t, J=10.0 Hz, 1H), 4.97-4.91 (m, 1H), 4.76 (s,2H), 4.36 (s, 1H), 3.12 (d, =8.9 Hz, 3H), 2.54 (s, 3H), 2.37 (s, 3H),2.05-1.97 (m, 1H), 1.97-1.93 (m, 3H), 1.49 (s, 3H), 1.41 (s, 3H), 1.09(d, J=3.5 Hz, 9H), 0.91 (tq, J=10.8, 4.9 Hz, 6H).

C₄₃H₅₈N₄O₅S calcd m/z=742.41 amu. found [M+H]⁺ =743.44, [M+Na]⁺=765.41.

Example 91

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(4-methoxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared according to General Procedure 9 from Bocprotected Example 86.

Major Diastereomer:

¹H NMR (400 MHz, Methanol-d₄) δ 7.44 (dd, J=12.9, 8.6 Hz, 2H), 7.40-7.34(m, 5H), 7.00 (t, J=8.4 Hz, 2H), 6.38 (d, J=9.2 Hz, 1H), 5.05 (t, J=9.9Hz, 1H), 4.93 (s, 1H), 4.75 (d, J=1.8 Hz, 2H), 4.29 (s, 1H), 3.84 (s,3H), 3.12 (s, 3H), 2.51 (s, 3H), 2.04-1.98 (m, 1H), 1.95 (d, J=1.4 Hz,3H), 1.45 (s, 3H), 1.37 (s, 3H), 1.09 (s, 9H), 0.92-0.86 (m, 6H).

Minor Diastereomer:

¹H NMR (400 MHz, Methanol-d₄) δ 7.44 (dd, J=12.9, 8.6 Hz, 2H), 7.40-7.34(m, 5H), 7.00 (t, J=8.4 Hz, 2H), 6.38 (d, J=9.2 Hz, 1H), 4.99 (t, J=10.1Hz, 1H), 4.93 (s, 1H), 4.75 (d, J=1.8 Hz, 2H), 4.26 (s, 1H), 3.82 (s,3H), 3.11 (s, 3H), 2.47 (s, 3H), 2.04-1.98 (m, 1H), 1.92 (d, f=1.4 Hz,3H), 1.53 (s, 3H), 1.48 (s, 3H), 0.94 (s, 9H), 0.92-0.86 (m, 6H).

C₃₅H₅₂N₄O₆S calcd m/z=656.36 amu. found [M+H]⁺ =657.35, [M+Na]⁺=679.25.

Example 92

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((R)-3-(3-methoxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared according to General Procedure 9 from Bocprotected(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(3-bromophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide.The two diastereomeric products resulted from diastereomerically impurestarting material and were separable by prep-scale HPLC.

Major Diastereomer:

¹H NMR (400 MHz, Methanol-d₄) δ 7.51-7.32 (m, 6H), 7.14-7.07 (m, 1H),7.06 (t, J=2.2 Hz, 1H), 6.98-6.90 (m, 1H), 6.38 (dd, J=9.6, 1.7 Hz, 1H),4.99 (t, J=10.3 Hz, 1H), 4.93 (s, 1H), 4.75 (d, J=1.8 Hz, 2H), 4.32 (s,1H), 3.85 (s, 3H), 3.11 (s, 3H), 2.47 (s, 3H), 2.04-1.96 (m, 1H), 1.93(d, J=1.4 Hz, 3H), 1.54 (s, 3H), 1.47 (s, 3H), 0.96 (s, 9H), 0.89 (dd,J=6.6, 3.4 Hz, 6H).

Minor diastereomer: refer to Example 93 (immediately following) for 1HNMR spectral data

C₃₅H₅₂N₄O₆S calcd m/z=656.36 amu. found [M+H]⁺ =657.36, [M+Na]⁺=679.29.

Example 93

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(3-methoxyphenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared according to Example 92. The twodiastereomeric products resulted from diastereomerically impure startingmaterial and were separable by prep-scale HPLC.

¹H NMR (400 MHz, Methanol-d₄) δ 7.39 (d, J=5.5 Hz, 6H), 7.11 (dd, =4.9,2.8 Hz, 3H), 6.38 (d, J=9.4 Hz, 1H), 5.06 (d, J=9.5 Hz, 1H), 4.93 (s,1H), 4.76 (s, 2H), 4.35 (s, 1H), 3.86 (s, 3H), 3.13 (s, 3H), 2.52 (s,3H), 2.05-1.97 (m, 1H), 1.95 (d, J=1.6 Hz, 3H), 1.46 (s, 3H), 1.38 (s,3H), 1.09 (s, 9H), 0.90 (t, J=6.6 Hz, 6H).

C₃₅H₅₂N₄O₆S calcd m/z=656.36 amu. found [M+H]⁺ =657.36, [M+Na]⁺=679.32.

Example 94

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-(4-(2-hydroxyethoxy)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

Title compound was prepared as follows: a mixture of Boc protectedExample 86, CuI (10 mol %), 3,4,7,8-tetramethyl-1,10-phenanthroline (20mol %), Cs₂CO₃ (2.5 eq), and ethylene glycol (90 eq) was stirred underN₂ at 130° C. for 20 h. The resulting mixture was diluted with H₂O,carefully acidified with 1M citric acid and extracted with CH₂Cl₂ (5×).The organics were combined, washed with brine (1×), dried over MgSO₄,filtered, concentrated in vacuo and purified via silica gel columnchromatography (eluted with AcOH/EtOAc/hexanes mixtures) to afford thecross-coupled product which was subsequently deprotected and purifiedaccording to General Procedure 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.46 (d, J=8.8 Hz, 2H), 7.38 (d, J=2.5Hz, 5H), 7.05 (d, J=8.4 Hz, 2H), 6.38 (d, J=9.5 Hz, 1H), 5.05 (t, J=10.1Hz, 1H), 4.93 (s, 1H), 4.76 (s, 2H), 4.28 (d, J=11.0 Hz, 1H), 4.13-4.04(m, 2H), 3.90 (t, J=4.6 Hz, 2H), 3.12 (d, J=6.2 Hz, 3H), 2.50 (d, J=16.9Hz, 3H), 2.05-1.97 (m, 1H), 1.94 (d, J=11.0 Hz, 3H), 1.56-1.34 (m, 6H),1.09 (s, 9H), 0.90 (t, J=6.4 Hz, 6H). C₃₆H₅₄N₄O₇S calcd m/z=686.37 amu.found [M+H]⁺=687.42, [M+Na]⁺=709.37.

Example 95

S-2-(4-((S)-4-((S)-1-(((S,E)-2,5-dimethyl-6-oxo-6-(benzylsulfonamido)hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-ylamino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)phenoxy)ethylethanethioate

Title compound was prepared as follows: Tributylphosphine (6 eq) wasadded to a cold (0° C.) stirring solution of di-tert-butylazodicarboxylate (6 eq) in THE After 0.5 h, a solution of the Bocprotected Example 94 (1 eq) in THF was added, followed by a solution ofAcSH (4.5 eq) in THF. The pale yellow mixture was stirred at 0° C. for 1h then at ambient temperature for 23 h. The resulting mixture wasconcentrated in vacuo, dissolved in EtOAc and successively washed with1M HCl (2×), sat′d NH₄Cl (1×) and brine (1×). The organics were driedover MgSO₄, filtered, concentrated in vacuo and purified via silica gelcolumn chromatography (eluted with AcOH/EtOAc/hexanes mixtures) toafford the Boc-protected thioacetate product (HPLC/MS—[M+Na]⁺=867.47).

The thioacetate was dissolved in CH₂Cl₂ and treated with TFA. Afterstirring for 1 h, the reaction mixture was concentrated in vacuo. Theyellow/brown residue was dissolved in minimal amount of CH₂Cl₂, cooledto 0° C. and treated with ether to precipitate out the desiredaminothioacetate as an off-white solid in 10° A) yield over twosynthetic steps.

¹H NMR (400 MHz, Methanol-d₄) δ 7.46 (d, J=8.7 Hz, 2H), 7.38 (d, J=2.4Hz, 5H), 7.03 (d, J=8.6 Hz, 2H), 6.38 (d, J=9.5 Hz, 1H), 5.05 (t, J=10.0Hz, 1H), 4.93 (s, 1H), 4.75 (s, 2H), 4.27 (d, J=11.4 Hz, 1H), 4.14 (t,=6.6 Hz, 2H), 3.28 (t, J=6.6 Hz, 2H), 3.11 (d, J=6.6 Hz, 3H), 2.49 (d,J=15.5 Hz, 3H), 2.38 (s, 3H), 2.05-1.97 (m, 1H), 1.95 (s, 3H), 1.45 (s,3H), 1.37 (s, 3H), 1.08 (s, 9H), 0.96-0.85 (m, 6H).

C₃₈H₅₆N₄O₇S₂ calcd m/z=744.36 amu. found [M+H]⁺=745.39, [M+Na]⁺=777.32.

Example 96

(S,E)-4-((S)-2-((S)-3-(4-(2-aminoethoxy)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide

Title compound was prepared as follows: Et₃N (4 eq) was added to a cold(0° C.) stirring solution of MsCl (3.7 eq) in CH₂Cl₂. After 2 min, asolution of the Boc protected Example 94 in CH₂Cl₂ was added. The paleyellow mixture was stirred cold for 5 min and then at ambienttemperature for 72 h. The resulting mixture was dilute with EtOAc andsuccessively washed with 1M citric acid (1×), 1M NaHCO₃ (1×) and brine(1×). The organics were dried over MgSO₄, filtered and concentrated invacuo to afford the mesylated-alcohol (HPLC/MS—[M+Na]⁺=887.42) which wasused in the next step without further purification.

The mesylate was dissolved in DMF and treated with NaN₃ (7 eq). Theresulting suspension was stirred at ambient temperature for 18 h andthen at 60° C. for 5 h. The reaction mix was diluted with H₂O, acidifiedwith 1M HCl and extracted with CH₂Cl₂ (4×). The combined organics weredried over MgSO₄, filtered and concentrated in vacuo to afford the azidoproduct (HPLC/MS—[M+Na]⁺=834.44) which was used in the next step withoutfurther purification.

The azide was dissolved in THF/H₂O (10:1) and treated withtributylphosphine (3.5 eq). The mixture was stirred at ambienttemperature for 21 h and then concentrated in vacuo. The resultingresidue was dissolved in EtOAc and successively washed with 1M HCl (3×),1M NaHCO₃ (3×), H₂O (2×) and brine (2×). The organics were dried overMgSO₄, filtered, concentrated in vacuo and purified via silica gelcolumn chromatography (eluted with MeOH/CH₂Cl₂ mixtures) to afford theprimary amine as a white solid (HPLC/MS—[M+H]⁺=786.45).

The amine was dissolved in CH₂Cl₂ and treated with TFA. After stirringfor 1 h, the reaction mixture was concentrated in vacuo. The off-whitesolid residue was dissolved in minimal amount of MeOH, cooled to 0° C.and treated with ether to precipitate out the desired diamine product asan off-white solid in 6% yield over four synthetic steps.

¹H NMR (400 MHz, Methanol-d₄) δ 7.50 (d, J=8.6 Hz, 2H), 7.37 (s, 5H),7.09 (d, J=8.6 Hz, 2H), 6.41 (d, J=9.4 Hz, 1H), 5.02 (t, J=10.0 Hz, 1H),4.91 (s, 1H), 4.70 (s, 2H), 4.27 (t, J=5.0 Hz, 2H), 3.40 (t, J=5.0 Hz,2H), 3.37 (s, 1H), 3.12 (s, 3H), 2.47 (s, 3H), 2.06-1.95 (m, 1H), 1.94(d, J=1.4 Hz, 3H), 1.45 (s, 3H), 1.37 (s, 3H), 1.08 (s, 9H), 0.89 (dd,J=9.7, 6.6 Hz, 6H).

C₃₈H₅₅N₅O₆S calcd m/z=685.39 amu. found [M+H]⁺=686.32, [M+Na]⁺=708.27,[(M+2H)/2]²⁺=343.77.

Example 97

(S,E)-2,5-dimethyl-N-(2-(2,2,2-trifluoroacetamido)phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 an2,2,2-trifluoro-N-(2-sulfamoylphenyl)acetamide according to GeneralProcedures 2, and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.27 (d, J=8.4 Hz, 1H), 8.05 (d, J=7.8Hz, 1H), 7.67 (t, J=7.9 Hz, 1H), 7.54 (d, J=8.1 Hz, 2H), 7.48 (t, J=7.7Hz, 2H), 7.40 (dt, J=13.3, 7.4 Hz, 2H), 6.57 (d, J=9.2 Hz, 1H), 4.92 (s,2H), 4.34 (s, 1H), 3.17 (s, 3H), 2.50 (s, 3H), 2.06 (m, 1H), 1.87 (d,J=1.3 Hz, 3H), 1.45 (s, 3H), 1.33 (s, 3H), 1.07 (s, 9H), 0.91 (dd,J=6.6, 3.5 Hz, 6H).

¹⁹F NMR (377 MHz, Methanol-d₄) δ −76.96, −77.73. C₃₅H₄₈F₃N₅O₆S calcdm/z=723.33 amu. found [M+H]⁺=723.34, [M+Na]⁺=746.23.

Example 98

(S,E)-N-(2-aminophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 an2,2,2-trifluoro-N-(2-sulfamoylphenyl)acetamide according to GeneralProcedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.75 (dd, J=8.2, 1.5 Hz, 1H), 7.55 (d,J=7.8 Hz, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.38 (t, J=7.4 Hz, 1H), 7.33-7.27(m, 1H), 6.81 (d, J=8.2 Hz, 1H), 6.69 (t, J=7.5 Hz, 1H), 6.49 (dd,J=9.1, 1.5 Hz, 1H), 4.97 (t, J=10.1 Hz, 1H), 4.92 (s, 1H), 4.35 (s, 1H),3.17 (s, 3H), 2.51 (s, 3H), 2.07 (m, 1H), 1.88 (d, J=1.4 Hz, 3H), 1.46(s, 3H), 1.36 (s, 3H), 1.06 (s, 9H), 0.92 (t, J=6.8 Hz, 6H).

C₃₃H₄₉N₅O₅S calcd m/z=627.35 amu. found [M+H]⁺=628.36, [M+Na]⁺=650.37,[(M+2H)/2]²⁺=314.76.

Example 99

(S,E)-N-(biphenyl-4-ylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared using from Boc protected Example 56 withphenylboronic acid according to General Procedures 8 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.12 (d, J=8.3 Hz, 2H), 7.83 (d, J=8.4Hz, 2H), 7.71 (d, J=7.7 Hz, 2H), 7.52 (dd, J=11.6, 7.6 Hz, 4H), 7.45 (t,J=7.3 Hz, 3H), 7.36 (t, J=7.2 Hz, 1H), 6.52 (d, J=9.4 Hz, 1H), 4.96 (t,J=9.5 Hz, 1H), 4.92 (s, 1H), 4.33 (s, 1H), 3.18 (s, 3H), 2.50 (s, 3H),2.14-2.03 (m, 1H), 1.88 (s, 3H), 1.45 (s, 3H), 1.35 (s, 3H), 1.07 (s,9H), 0.92 (t, J=6.9 Hz, 6H).

C₃₉H₅₂N₄O₅S calcd m/z=688.37 amu. found [M+H]⁺=689.10, [M+Na]⁺=711.32.

Example 100

(S,E)-N-(4′-aminobiphenyl-4-ylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Boc protected Example 68 with4-(tert-butoxycarbonylamino)phenylboronic acid according to GeneralProcedures 8 and 7

¹H NMR (400 MHz, Methanol-d₄) δ 8.05 (d, J=8.6 Hz, 2H), 7.75 (d, =8.6Hz, 2H), 7.59-7.51 (m, 4H), 7.45 (t, J=7.7 Hz, 2H), 7.36 (t, J=7.3 Hz,1H), 6.91 (d, J=8.3 Hz, 2H), 6.50 (d, J=9.1 Hz, 1H), 4.98-4.92 (m, 1H),4.91 (s, 1H), 4.34 (s, 1H), 3.18 (s, 3H), 2.50 (s, 3H), 2.13-2.03 (m,1H), 1.88 (d, J=1.4 Hz, 3H), 1.45 (s, 3H), 1.35 (s, 3H), 1.06 (s, 9H),0.92 (t, J=6.2 Hz, 6H).

C₃₉H₅₃N₅O₅S calcd m/z=703.38 amu. found [M+H]⁺=704.26, [M+Na]⁺=726.41,[(M+2H)/2]²⁺=352.77.

Example 101

(S,E)-N-(4-fluorobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 4-fluorobenzylsulfonamideaccording to General Procedures 2 and 7.

¹H NMR (400 MHz, Methanol-d4) δ 7.60-7.52 (m, 2H), 7.48 (t, J=7.7 Hz,2H), 7.44-7.34 (m, 3H), 7.18-7.05 (m, 2H), 6.41 (dd, J=9.5, 1.7 Hz, 1H),5.06 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.74 (s, 2H), 4.35 (s, 1H), 3.13(s, 3H), 2.51 (s, 3H), 2.07-1.97 (m, 1H), 1.95 (d, J=1.4 Hz, 3H), 1.48(s, 3H), 1.39 (s, 3H), 1.09 (s, 9H), 0.90 (t, J=6.3 Hz, 6H).

C₃₄H₄₉FN₄O₅S calcd m/z=644.34. found [M+H]⁺ =645.32.

Example 102

(S,E)-2,5-dimethyl-N-(3-(trifluoromethyl)benzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and3-trifluorobenzylsulfonamide according to General Procedures 2 and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.74-7.64 (m, 3H), 7.61 (d, J=7.7 Hz,1H), 7.60-7.54 (m, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.38 (t, J=7.3 Hz, 1H),6.42 (dd, J=9.4, 1.7 Hz, 1H), 5.06 (t, J=10.0 Hz, 1H), 4.93 (s, 1H),4.36 (s, 1H), 3.13 (s, 3H), 2.51 (s, 3H), 2.07-1.97 (m, 1H), 1.95 (d,J=1.4 Hz, 3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.08 (s, 9H), 0.89 (d, J=6.5Hz, 6H).

C₃₅H₄₉F₃N₄O₅S calcd m/z=694.34. found [M+H]⁺=695.38.

Example 103

(S,E)-2,5-dimethyl-N-(3-(trifluoromethoxy)benzylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and3-trifluoromethoxybenzylsulfonamide according to General Procedures 2,and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.56 (d, J=7.8 Hz, 2H), 7.48 (t, J=7.9Hz, 3H), 7.43-7.36 (m, 2H), 7.32 (d, J=9.3 Hz, 2H), 6.43 (dd, J=9.4, 1.7Hz, 1H), 5.06 (t, J=10.0 Hz, 1H), 4.93 (s, 1H), 4.82 (s, 2H), 4.35 (s,1H), 3.13 (s, 3H), 2.51 (s, 3H), 2.07-1.97 (m, 1H), 1.95 (d, J=1.4 Hz,3H), 1.48 (s, 3H), 1.39 (s, 3H), 1.08 (s, 9H), 0.90 (dd, J=6.6, 4.3 Hz,6H).

C₃₅H₄₉F₃N₄O₆S calcd m/z=710.33. found [M+H]⁺ =711.38.

Example 104

(S,E)-N-(3,4-dichlorobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and3,4-dichlorobenzylsulfonamide according to General Procedures 2, and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.56 (td, J=5.2, 4.5, 1.9 Hz, 4H), 7.48(t, J=7.7 Hz, 2H), 7.38 (t, J=7.3 Hz, 1H), 7.33 (dd, J=8.4, 2.1 Hz, 1H),6.41 (dd, J=9.5, 1.8 Hz, 1H), 5.06 (t, J=10.0 Hz, 1H), 4.93 (s, 1H),4.77 (s, 2H), 4.36 (s, 1H), 3.14 (s, 3H), 2.52 (s, 3H), 2.07-1.97 (m,1H), 1.95 (d, J=1.4 Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.08 (s, 9H),0.90 (dd, J=6.6, 4.9 Hz, 6H).

C₃₄H₄₈Cl₂N₄O₅S calcd m/z=694.27. found [M+H]⁺ =695.32.

Example 105

(S,E)-N-(2-cyanobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 2-cyanobenzylsulfonamideaccording to General Procedures 2, and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.81 (dd, J=7.7, 1.3 Hz, 1H), 7.72 (td,J=7.7, 1.3 Hz, 1H), 7.66 (d, J=7.7 Hz, 1H), 7.62-7.59 (m, 1H), 7.58-7.53(m, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.38 (t, J=7.3 Hz, 1H), 6.50 (d, J=9.4Hz, 1H), 5.08 (dd, J=10.6, 9.3 Hz, 1H), 4.99 (s, 2H), 4.95 (s, 1H), 4.36(s, 1H), 3.16 (s, 3H), 2.52 (s, 3H), 2.09-1.99 (m, 1H), 1.98 (d, J=1.4Hz, 3H), 1.49 (s, 3H), 1.39 (s, 3H), 1.10 (s, 9H), 0.94 (d, J=6.6 Hz,3H), 0.91 (d, J=6.6 Hz, 3H).

C₃₅H₄₉N₅O₅S calcd m/z=651.35. found [M+H]⁺ =652.38.

Example 106

(S,E)-N-(3-chlorobenzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 3-chlorobenzylsulfonamideaccording to General Procedures 2, and 7.

1H NMR (400 MHz, Methanol-d4) δ 7.58-7.53 (m, 2H), 7.48 (t, J=7.6 Hz,2H), 7.43-7.34 (m, 4H), 7.32 (d, J=7.5 Hz, 1H), 6.42 (d, J=9.5 Hz, 1H),5.06 (t, J=10.0 Hz, 1H), 4.94 (s, 1H), 4.74 (s, 2H), 4.33 (s, 1H), 3.13(s, 3H), 2.50 (s, 3H), 2.07-1.97 (m, 1H), 1.95 (d, J=1.4 Hz, 3H), 1.48(s, 3H), 1.39 (s, 3H), 1.08 (s, 9H), 0.90 (t, J=7.2 Hz, 6H).

C₃₄H₄₉C1N₄O₅S calcd m/z=660.31. found [M+H]⁺=661.32.

Example 107

(S,E)-N-(4-amino-2-ethylphenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and 2-ethylbenzylsulfonamideaccording to General Procedures 2, and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.79 (d, J=8.7 Hz, 1H), 7.55 (d, J=7.9Hz, 2H), 7.48 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.4 Hz, 1H), 6.57 (d, J=2.3Hz, 1H), 6.54 (dd, J=8.8, 2.4 Hz, 1H), 6.46 (d, J=9.4 Hz, 1H), 5.01 (t,J=10.0 Hz, 1H), 4.92 (s, 1H), 4.34 (s, 1H), 3.16 (s, 3H), 2.99-2.90 (m,2H), 2.50 (s, 3H), 2.11-2.00 (m, 1H), 1.87 (d, J=1.4 Hz, 3H), 1.47 (s,3H), 1.38 (s, 3H), 1.22 (t, J=7.5 Hz, 3H), 1.06 (s, 9H), 0.91 (dd, J=6.6Hz, 6H).

C₃₅H₅₃N₅O₅S calcd m/z=655.38. found [M+H]⁺ =656.4.

Example 108

(S,E)-N-(4-amino-3-(trifluoromethoxy)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(4-sulfamoyl-2-(trifluoromethoxy)phenyl)acetamideaccording to General Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.81-7.75 (m, 1H), 7.71 (dd, J=8.7, 2.1Hz, 1H), 7.55 (d, J=7.9 Hz, 2H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.1Hz, 1H), 6.89 (d, J=8.7 Hz, 1H), 6.51-6.42 (m, 1H), 4.98 (t, J=10.0 Hz,1H), 4.92 (t, J=4.1 Hz, 1H), 4.37 (s, 1H), 3.16 (s, 3H), 2.51 (s, 3H),2.12-2.01 (m, 1H), 1.88 (d, J=1.4 Hz, 3H), 1.47 (s, 3H), 1.37 (s, 3H),1.07 (s, 9H), 0.92 (dd, J=6.6 Hz, 6H).

C₃₄H₄₈F₃N₅O₆S calcd m/z=711.33. found [M+H]⁺=712.4.

Example 109

(S,E)-N-(4-amino-2,3-dimethylphenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(4-sulfamoyl-2,3-dimethylphenyl)acetamide according toGeneral Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.75 (d, J=8.8 Hz, 1H), 7.55 (d, =7.9Hz, 2H), 7.47 (t, J=7.7 Hz, 2H), 7.37 (t, J=6.9 Hz, 1H), 6.63 (d, =8.8Hz, 1H), 6.46 (d, J=9.7 Hz, 1H), 5.00 (t, J=10.0 Hz, 1H), 4.93 (s, 1H),4.32 (s, 1H), 3.17 (s, 3H), 2.54 (s, 3H), 2.49 (s, 3H), 2.09 (s, 3H),2.08-2.02 (m, 1H), 1.87 (d, J=1.4 Hz, 3H), 1.47 (s, 3H), 1.37 (s, 3H),1.07 (s, 9H), 0.92 (dd, J=6.8, 6.5 Hz, 6H).

C₃₅H₅₃N₅O₅S calcd m/z=655.38. found [M+H]⁺=656.4.

Example 110

(S,E)-N-(4-amino-5,6,7,8-tetrahydronaphthalen-1-ylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(4-sulfamoyl-5,6,7,8-tetrahydronaphthalen-1-yl)acetamideaccording to General Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.74 (d, J=8.7 Hz, 1H), 7.55 (d, J=7.9Hz, 2H), 7.48 (t, J=7.6 Hz, 2H), 7.38 (t, J=7.2 Hz, 1H), 6.60 (d, J=8.7Hz, 1H), 6.46 (d, J=9.2 Hz, 1H), 5.00 (t, J=10.0 Hz, 1H), 4.95-4.91 (m,1H), 4.36 (s, 1H), 3.17 (s, 3H), 3.10-3.05 (m, 2H), 2.51 (s, 3H), 2.46(t, J=6.5 Hz, 2H), 2.10-2.02 (m, 1H), 1.88 (s, 3H), 1.87-1.75 (m, 4H),1.47 (s, 3H), 1.38 (s, 3H), 1.07 (s, 9H), 0.92 (dd, J=7.1 Hz, 6H).

C₃₇H₅₅N₅O₅S calcd m/z=681.39. found [M+H]⁺=682.4.

Example 111

(S,E)-N-(4-amino-3-methylphenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(2-methyl-4-sulfamoylphenyl)acetamide according toGeneral Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.64 (s, 1H), 7.61 (dd, J=8.5, 2.3 Hz,1H), 7.57-7.51 (m, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.41-7.35 (m, 1H), 6.71(d, J=8.5 Hz, 1H), 6.43 (dd, J=9.3, 1.6 Hz, 1H), 4.96 (t, J=10.0 Hz,1H), 4.92 (s, 1H), 4.35 (s, 1H), 3.16 (s, 3H), 2.51 (s, 3H), 2.17 (s,3H), 2.10-2.01 (m, 1H), 1.87 (d, J=1.4 Hz, 3H), 1.46 (s, 3H), 1.36 (s,3H), 1.07 (s, 9H), 0.91 (dd, J=6.3 Hz, 6H).

C₃₄H₅₁N₅O₅S calcd m/z=641.36. found [M+H]⁺ =642.4.

Example 112

(S,E)-N-(4-amino-3-fluorophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(2-fluoro-4-sulfamoylphenyl)acetamide according toGeneral Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d⁴) δ 7.62-7.55 (m, 3H), 7.54 (s, 1H), 7.48(t, J=7.7 Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 6.85 (t, J=8.6 Hz, 1H), 6.45(d, J=9.3 Hz, 1H), 4.98 (t, J=9.9 Hz, 1H), 4.92 (s, 1H), 4.34 (s, 1H),3.16 (s, 3H), 2.50 (s, 3H), 2.12-2.00 (m, 1H), 1.88 (d, J=1.4 Hz, 3H),1.46 (s, 3H), 1.37 (s, 3H), 1.07 (s, 9H), 0.91 (dd, J=6.8 Hz, 6H).

C₃₃H₄₈FN₅O₅S calcd m/z=645.34. found [M+H]⁺ =646.4.

Example 113

(S,E)-N-(4-amino-3-ethylphenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(2-ethyl-4-sulfamoylphenyl)acetamide according toGeneral Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.66 (d, J=2.3 Hz, 1H), 7.61 (dd, J=8.6,2.3 Hz, 1H), 7.55 (d, J=7.6 Hz, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.37 (t,J=7.3 Hz, 1H), 6.71 (d, J=8.5 Hz, 1H), 6.43 (dd, J=9.3, 1.7 Hz, 1H),4.96 (t, J=9.9 Hz, 1H), 4.92 (s, 1H), 4.35 (s, 1H), 3.16 (s, 3H), 2.54(dd, J=7.4, 2.2 Hz, 2H), 2.51 (s, 3H), 2.12-1.99 (m, 1H), 1.87 (d, J=1.4Hz, 3H), 1.46 (s, 3H), 1.36 (s, 3H), 1.27 (t, J=7.5 Hz, 3H), 1.07 (s,9H), 0.91 (dd, J=6.4 Hz, 6H)

C₃₅H₅₃N₅O₅S calcd m/z=655.38. found [M+H]⁺=656.5.

Example 114

(S,E)-N-(4-amino-3-(trifluoromethyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide

Title compound was prepared from Example 3 and2,2,2-trifluoro-N-(2-trifluoromethyl-4-sulfamoylphenyl)acetamideaccording to General Procedures 2, 3 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 8.04 (s, 1H), 7.87 (d, J=8.8 Hz, 1H),7.55 (d, J=7.6 Hz, 2H), 7.48 (t, J=7.3 Hz, 2H), 7.36 (dd, J=14.5, 7.4Hz, 1H), 6.89 (d, J=8.9 Hz, 1H), 6.47 (d, J=9.3 Hz, 1H), 4.99 (t, J=10.2Hz, 1H), 4.92 (s, 1H), 4.33 (s, 1H), 3.16 (s, 3H), 2.50 (s, 3H),2.11-2.00 (m, 1H), 1.88 (s, 3H), 1.47 (s, 3H), 1.37 (s, 3H), 1.07 (s,9H), 0.91 (dd, J=7.0 Hz, 6H).

C₃₄H₄₈F₃N₅O₅S calcd m/z=695.33. found [M+H]⁺ =696.4.

Example 116

S)—N—((S)-1-((S)-2-((E)-3-(3-mercaptopropylsulfonamido)-2-methyl-3-oxoprop-1-enyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3-methyl-2-(methylamino)-3-phenylbutanamide

The title compound was synthesized from Boc-proline and Example 2according to General Procedures 10, 11, 2, 3, 7 and others from NiemanJ. A. et al. J. Nat. Prod. 2003, 66, 183-199. The compound was isolatedas two diastereoisomers in an approximately 1:1 ratio.

¹H NMR (400 MHz, Methanol-d₄) δ 7.57-7.12 (m, 5H), 6.39 (dd, J=9.4, 1.6Hz, 0.5H), 6.31 (dd, J=8.2, 1.5 Hz, 0.5H), 4.72 (q, J=7.5 Hz, 0.5H),4.66-4.56 (m, 0.5H), 4.40 (s, 0.5H), 4.28 (d, J=11.9 Hz, 1H), 3.81 (m,0.5H), 3.76-3.56 (m, 3H), 2.77-2.64 (m, 2H), 2.59 (m, 3H), 2.39-2.22 (m,1H), 2.18-1.72 (m, 7H), 1.61-1.33 (m, 6H), 1.15-0.85 (m, 11H).

C₂₉H₄₆N₄O₅S₂ calcd m/z=594.35. found [M+H]⁺=595.3.

Example 117

(S)—N—((S)-1-(2-(3-(3-mercaptopropylsulfonamido)-2-methyl-3-oxoprop-1-enyl)piperidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3-methyl-2-(methylamino)-3-phenylbutanamide

The title compound was synthesized from Boc-homoproline and Example 2according to General Procedures 10, 11, 2, 3, 7 and others from NiemanJ. A. et al. J. Nat. Prod. 2003, 66, 183-199. The compound was isolatedas two diastereoisomers in an approximately 2:3 ratio.

¹H NMR (600 MHz, Methanol-d₄) δ 7.55 (d, J=7.8 Hz, 1H), 7.46 (m, 3H),7.38 (m, 1H), 6.81 (d, J=8.3 Hz, 0.6H), 6.79 (d, J=7.8 Hz, 0.4H), 5.66(m, 0.6H), 5.12 (m, 0.4H), 5.05 (s, 0.6H), 4.86 (s, 0.4H), 4.42 (d,J=14.9 Hz, 0.4H), 4.35 (s, 0.6H), 4.26 (s, 0.4H), 4.12 (d, J=13.8 Hz,0.6H), 3.64 (d, J=7.6 Hz, 1H), 3.63 (d, J=7.4 Hz, 1H), 3.39 (m, 0.6H),2.94 (td, J=13.8, 2.6 Hz, 0.4H), 2.68 (t, J=6.7 Hz, 2H), 2.56 (m, 3H),2.10 (m, 3.5H), 1.97 (s, 1.5H), 1.90-1.70 (m, 7H), 1.65-1.29 (m, 6H),1.07 (s, 3.5H), 1.04 (s, 4.5H) ppm.

C₃₀H₄₇N₄O₅S₂ calcd. m/z=608.31. found [M+H]⁺=609.32.

Example 118

(S)—N—((S)-1-(2-(3-(4-(mercaptomethyl)phenylsulfonamido)-2-methyl-3-oxoprop-1-enyl)piperidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3-methyl-2-(methylamino)-3-phenylbutanamide

The title compound was synthesized from Boc-homoproline and Example 7according to General Procedures 10, 11, 2, 3, 7 and others from NiemanJ. A. et al. J. Nat. Prod. 2003, 66, 183-199. The compound was isolatedas two diastereoisomers in an approximately 2:3 ratio.

¹H NMR (600 MHz, Methanol-d₄) δ 8.02 (d, J=8.4 Hz, 0.8H), 8.00 (d, J=8.5Hz, 1.2H), 7.58 (d, J=8.5 Hz, 1H), 7.54 (d, J=8.5 Hz, 2H), 7.45 (t,J=8.2 Hz, 2H), 7.40 (d, J=7.2 Hz, 0.6H), 7.36 (m, 1H), 7.31 (t, J=7.1Hz, 0.4H), 6.74 (d, J=8.2 Hz, 1H), 5.59 (m, 0.6H), 5.06 (m, 0.4H), 5.02(s, 0.6H), 4.84 (s, 0.4H), 4.39 (d, J=12.5 Hz, 0.4H), 4.34 (s, 0.6H),4.20 (s, 0.4H), 4.08 (d, J=12.0 Hz, 0.6H), 3.83 (s, 1.2H), 3.73 (s,0.8H), 3.35 (m, 0.6H), 2.93 (td, J=13.6, 3.0 Hz, 0.4H), 2.55 (m, 3H),2.00 (s, 1H), 1.90-1.51 (m, 7H), 1.51-1.30 (m, 4H), 1.30 (s, 1H), 1.15(s, 1H), 1.04 (s, 3.5H), 1.01 (s, 4.5H) ppm.

C₃₄H₄₇N₄O₅S₂ calcd. m/z=656.31. found [M+H]⁺ =657.30.

Example 119

The title compound was prepared by application of general procedures 15and 7 from from Boc protected Example 77.

¹H NMR (400 MHz, Methanol-d₄) δ 7.58 (d, J=8.2 Hz, 2H), 7.49 (d, J=7.5Hz, 2H), 7.38 (t, J=7.7 Hz, 2H), 7.36-7.24 (m, 6H), 7.22 (d, J=7.8 Hz,2H), 6.81 (s, 2H), 6.57 (d, J=9.1 Hz, 1H), 5.08 (s, 2H), 5.04 (t, J=10.0Hz, 1H), 4.91 (s, 1H), 4.53 (dd, J=9.0, 5.1 Hz, 1H), 4.40 (s, 2H), 4.28(s, 2H), 4.19 (d, J=7.4 Hz, 1H), 3.49 (t, J=7.1 Hz, 2H), 3.26-3.11 (m,2H), 3.07-2.93 (m, 3H), 2.30 (t, J=7.4 Hz, 2H), 2.18 (s, 3H), 2.15-2.05(m, 1H), 1.99-1.91 (m, 1H), 1.89 (s, 3H), 1.83-1.72 (m, 1H), 1.72-1.53(m, 7H), 1.44 (s, 3H), 1.37 (s, 3H), 1.35-1.27 (m, 2H), 1.03 (s, 9H),1.00 (d, J=6.8 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H), 0.88 (d, J=6.5 Hz, 3H),0.82 (d, J=6.6 Hz, 3H).

C₆₄H₉₁N₁₁O₁₃S calcd. m/z=1253.7. found [M+H]⁺=1254.8.

Example 120

4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl4-(N—((S,E)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzylcarbamate

MC-VC-PABC-85

The title compound was prepared by application of general procedures 15and 7 to Boc protected Example 85.

C₆₃H₈₉N₁₁O₁₃S calcd. m/z=1239.6. found [M+H]⁺=1240.9.

Example 121

The title compound was prepared by application of general procedures 15and 7 to Boc protected Example 80.

C₆₃H₈₉N₁₁O₁₃S calcd. m/z=1239.6. found [M+H]⁺ =1240.9.

Example 122

The title compound was prepared by application of General Procedure 15to Example 41.

C₆₄H₉₁N₁₁O₁₃S calcd. m/z=1253.65. found [M+H]⁺ =1254.75,[M+2H]²⁺=628.20.

Example 123

(R)—N-(benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hexanamide

A suspension of the Example 14 and 10% palladium on carbon (25 mol % Pd)in glacial acetic acid was stirred under a H₂ atmosphere (1 atm) atambient temperature. After 142 h, the reaction suspension was passedthrough a bed of celite, rinsed with MeOH (5×) and concentrated invacuo. The residual light brown crude film was dissolved and purified onthe preparative HPLC (30-70% MeCN/H₂O with 0.1% TFA) and lyophilized toafford one diastereomer of the reduced product as a pale yellow solid in15% yield

¹H NMR (400 MHz, Methanol-d₄) δ 7.55 (d, J=7.2 Hz, 2H), 7.46 (t, J=7.8Hz, 2H), 7.43-7.31 (m, 6H), 5.01 (s, 1H), 4.79 (d, J=14.1 Hz, 1H), 4.65(d, J=14.1 Hz, 1H), 4.35 (s, 1H), 4.24 (s, 1H), 3.07 (s, 3H), 2.52 (s,3H), 2.27 (m, J=10.3, 7.0, 3.2 Hz, 1H), 2.14 (ddd, J=13.5, 10.6, 2.7 Hz,2H), 1.78 (d, J=8.6 Hz, 1H), 1.47 (s, 3H), 1.34 (s, 3H), 1.15 (d, J=6.9Hz, 3H), 1.14 (s, 9H), 1.04 (d, J=6.6 Hz, 3H), 0.82 (d, J=6.6 Hz, 3H).

C₃₄H₅₂N₄O₅S calcd m/z=628.37 amu. found [M+H]⁺ =629.6, [M+Na]⁺ =651.6.

General Synthetic Schemes for (T)-(L)-(D) Using LC-SPDP and SMCC Linkers

Composition produced using the SPDP linkage method described below. NoteR₂′ is distinct from R₂, as R₂ includes R₂′—S.

Composition produced using the SMCC linkage method described below. NoteR₂′ is distinct from R₂, as R₂ includes R₂′—S.

Composition produced using the SPDP linkage method described below. NoteR₁′ is distinct from R₁, as R₁ includes R₁′—S.

Composition produced using the SMCC linkage method described below. NoteR₁′ is distinct from R₁, as R₁ includes R₁′—S.

Example 124

(Compound A—SPDP—mAb) produced using the Compound A synthesis method,above, and the SPDP linkage method described below.

Example 125

(Compound B—SPDP—mAb) produced using the Compound B synthesis method,above, and the SPDP linkage method described below.

Example 126

(Compound C—SPDP—mAb) produced using the Compound C synthesis method,above, and the SPDP linkage method described below.

Example 127

(Compound B—SMCC—mAb) produced using the Compound B synthesis method,above, and the SMCC linkage method described below.

Example 128

(Compound A—SMCC—mAb) produced using the Compound A synthesis method,above, and the SMCC linkage method described below.

Example 129

(Compound C—SMCC—mAb) produced using the Compound C synthesis method,above, and the SMCC linkage method described below.

Example 130

To a vigorously stirred solution of bromobenzene (4.70 g, 30.0 mmol) and3,3-dimethylacrylic acid (1.00 g, 10.0 mmol) in 20 mL CH₂Cl₂ cooled to−10° C. in an NH₄Cl_((aq))/ice bath, solid AlCl₃ was added portion-wise,keeping the internal temperature below −5° C. The solution turnedyellow, then brown after addition. After one hour, analysis by LC andTLC indicated complete consumption of the limiting reagent. The reactionwas then quenched by the addition of 1 M citric acid, causing the browncolor to fade to yellow. The resulting sloppy suspension was extractedfour times with 20 mL Et₂O, the combined organics washed withNaCl_((sat)), dried over Na₂SO_(4(s)), and concentrated in vacuo withheating to 45° C. to remove solvent and residual bromobenzene. Theresulting oil solidified slowly. Recrystallization of the crude solid inhexanes afforded the title compound (1.29 g, 50%) as clusters of whiteprisms.

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.42 (d, J=8.6 Hz, 2H), 7.23 (d,J=8.6 Hz, 2H), 2.63 (s, 2H), 1.43 (s, 6H). C₁₁H₁₃BrO₂ calcd.[M+H]⁺=257.02 amu. found m/z=257.03. R_(f)=0.21 (20% (2%AcOH/EtOAc)/Hex).

Example 131

The title compound was prepared in the same manner as3-methyl-3-phenylbutanoic acid in Nieman J. A., et al. J Nat. Prod.2003, 66, 183-199, using bromobenzene in place of benzene as thesolvent, and substituting the acid-base workup with a simple extractionof the reaction mixture from 1 M citric acid and three successiverecrystallizations from hexanes. From a crude product enriched in thedesired meta isomer as a 2:1 mixture, the title compound could beobtained as white stubby needles in greater than 95% purity.

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.49 (t, J=1.9 Hz, 1H), 7.34(ddd, J=7.9, 1.9, 1.0 Hz, 1H), 7.29 (ddd, J=7.9, 1.9, 1.0 Hz, 1H), 7.18(t, J=7.9 Hz, 1H), 2.64 (s, 2H), 1.44 (s, 6H). C₁₁H₁₃BrO₂ calcd. [M+H]⁺=257.02 amu. found m/z=257.01. R_(f)=0.21 (20% (2% AcOH/EtOAc)/Hex).

Example 132

(S)-methyl3-(4-bromophenyl)-2-(tert-butoxycarbonyl(methyl)amino)-3-methylbutanoate

The title compound was synthesized from Example 130 according to thesequence of procedures described by Nieman et al. for the synthesis of(S)-methyl2-(tert-butoxycarbonyl(methyl)amino)-3-methyl-3-phenylbutanoate.

Example 133

(S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methylbutanoicacid

To a stirred solution of Example 68 (157 mg, 0.405 mmol) inpentaethylene glycol (1.5 mL) were added CsCO₃ (330 mg, 1.01 mmol),3,4,7,8-tetramethyl-1,10-phenanthroline (57 mg, 0.24 mmol), and CuI (23mg, 0.12 mmol). Nitrogen was blown into the flask, then it was sealedand heated to 130° C., the solution quickly turning red to brown toblack. After 40 h, the reaction looked to be nearly complete by HPLCanalysis. Thus, the mixture was allowed to cool to ambient temperature,diluted with H₂O, and transferred to a larger Erlenmeyer with a stirbar. This mixture was carefully acidified to pH ˜3 with 1 M citric acid,paying attention not to allow the foamy mixture to spill over. Themixture was then extracted five times with CH₂Cl₂, the combined organicextracts washed with NaCl_((sat)), dried over Na₂SO_(4(s)), andconcentrated in vacuo to yield about 300 mg of crude oil. Purificationby flash chromatography (1-10% MeOH/(2% AcOH/EtOAc)) yielded the titlecompound (66 mg, 30%) as a clear film which existed as a set of N-Bocrotamers an an approximate 2:1 ratio.

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.35 (d, J=7.8 Hz, 1.3H), 7.30(d, J=7.6 Hz, 0.7H), 6.87 (d, J=7.1 Hz, 2H), 5.07 (s, 0.7H), 4.93 (s,0.3H), 4.14 (m, 2H), 3.86 (m, 2H), 3.70 (m, 16H), 2.83 (s, 1H), 2.72 (s,2H), 1.54 (s, 3H), 1.49 (s, 3H), 1.45 (s, 9H). C₂₇H₄₅NO₁₀ calcd.[M+H]⁺=544.31 amu. found m/z=544.36. R_(f)=0.36 (5% MeOH/(2%AcOH/EtOAc)).

Example 134

(S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-(2-(2hydroxyethoxy)ethoxy)ethoxy)ethoxy)phenyl)-3-methylbutanoic acid

The title compound was prepared according to the above method fromExample 68 (132 mg, 0.341 mmol), CsCO₃ (278 mg, 0.853 mmol),3,4,7,8-tetramethyl-1,10-phenanthroline (24 mg, 0.10 mmol), and CuI (10mg, 0.051 mmol). Flash chromatography (1-10% MeOH/(2% AcOH/EtOAc)) gavethe title compound (66 mg, 38%) as a clear oil in an approximate 2:1ratio of N-Boc rotamers.

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.34 (d, J=8.4 Hz, 1.3H), 7.29(d, J=8.1 Hz, 0.7H), 6.85 (d, J=8.4 Hz, 2H), 5.05 (s, 0.7H), 4.91 (s,0.3H), 4.13 (t, J=4.6 Hz, 2H), 3.87-3.79 (m, 2H), 3.76-3.60 (m, 10H),3.59 (t, J=4.1 Hz, 2H), 2.80 (s, 1H), 2.69 (s, 2H), 1.53 (s, 3H), 1.48(s, 3H), 1.44 (s, 9H). C₂₅H₄₁NO₉ calcd. [M+H]⁺=500.29 amu. foundm/z=500.36. R_(f)=0.46 (5% MeOH/(2% AcOH/EtOAc)).

Example 135

(S)-3-(3-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methyl-2-(methylamino)butanoicacid

The precursor to the title compound,(S)-3-(3-bromophenyl)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoicacid, was prepared from Example 131 by following the procedures inNeiman et al.

Thus, following the procedures above, from(S)-3-(3-bromophenyl)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoicacid (166 mg, 0.43 mmol), CsCO₃ (330 mg, 1.01 mmol),3,4,7,8-tetramethyl-1,10-phenanthroline (31 mg, 0.13 mmol), and CuI(12.3, 0.060 mmol) in 1.5 mL pentaethylene glycol heated to 130° C. fortwo days, the title compound (73 mg, 31%) was obtained as a clear oilafter flash chromatography (1-10% MeOH/(2% AcOH/EtOAc)) in anapproximate 2:1 ratio of N-Boc rotamers.

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.17 (t, J=7.8 Hz, 1H), 7.14-7.07(m, 1H), 7.07-6.93 (m, 2H), 6.74 (d, 0.1=8.0 Hz, 1H), 5.11 (s, 0.7H),4.93 (s, 0.3H), 4.25-4.03 (m, 2H), 3.91-3.77 (m, 2H), 3.78-3.66 (m, 2H),3.69-3.43 (s, 14H), 2.72 (s, 1H), 2.65 (s, 1H), 1.51 (s, 3H), 1.49 (s,3H), 1.45 (s, 9H). C₂₇H₄₅NO₁₀ calcd. [M+H]⁺=544.31 amu. foundm/z=544.34.

Example 136

(6S,9S,12S,E)-ethyl9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4-((16-oxo-3,6,9,12-tetraoxa-15-thiaheptadecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate

(S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methylbutanoicacid (65 mg, 0.120 mmol) was coupled to (S,E)-ethyl4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate withHATU and DIPEA following the same stoichiometry and procedure asdescribed in the general coupling procedures in Nieman et al. to give anintermediate free alcohol after purification by flash chromatography(1-10% MeOH/(2% AcOH/EtOAc)). Next, to triphenylphosphine (40 mg, 0.15mmol) in 0.75 mL THF under N₂ at 0° C., di-tert-butylazodicarboxylate(35 mg, 0.15 mmol) was added in one portion. After 35 minutes, a whiteprecipitate crashed out and the reaction became difficult to stir. Tothis suspension, a solution of the intermediate alcohol (42 mg, 0.050mmol) in 0.75 mL THF was added diluting the precipitate enough torestore stirring. Five minutes later, thioacetic acid (5.7 mg, 0.075mmol) in 0.05 mL THF was added causing all yellow color to fade from themixture. After 30 min, the reaction was allowed to warm to ambienttemperature. The precipitate disappeared after another 15 min, andanalysis by TLC and LCMS showed nearly complete conversion. Afteranother 40 minutes, the reaction mixture was concentrated in vacuo, thensubjected directly to flash chromatography (40-100% EtOAc/Hex then to10% MeOH/EtOAc) to yield the title compound (26 mg, 57%) as a clearfilm.

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.43 (d, J=8.4 Hz, 1.3H), 7.31(d, J=8.3 Hz, 0.7H), 6.97-6.72 (m, 2H), 6.62 (dd, J=9.3, 1.6 Hz, 1H),6.14 (d, J=9.6 Hz, 1H), 5.22 (s, 0.7H), 5.12-4.99 (m, 1H), 4.84 (s,0.3H), 4.69 (d, J=9.3 Hz, 0.3H), 4.60 (d, J=8.9 Hz, 0.7H), 4.19 (q,J=7.2 Hz, 2H), 4.09 (td, J=4.6, 2.3 Hz, 2H), 3.84 (t, J=4.9 Hz, 2H),3.77-3.70 (m, 2H), 3.70-3.61 (m, 10H), 3.59 (t, J=6.4 Hz, 2H), 3.07 (t,J=6.4 Hz, 2H), 2.97-2.91 (m, 3H), 2.84 (s, 3H), 2.32 (s, 3H), 1.87 (s,3H), 1.49 (s, 3H), 1.43 (s, 9H), 1.35 (s, 3H), 1.30 (t, J=7.1 Hz, 3H),0.87 (d, J=6.6 Hz, 3H), 0.80 (d, J=16.6 Hz, 3H), 0.77 (s, 9H).C₄₆H₇₇N₃O₁₂S calcd. [M+H]⁺=896.53 amu. found m/z=896.77. R_(f)=0.56 (80%EtOAc/Hex).

Example 137

(6S,9S,12S,E)-ethyl9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4-((13-oxo-3,6,9-trioxa-12-thiatetradecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate

The title compound was prepared from(S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-(4-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethoxy)phenyl)-3-methylbutanoic acid (66 mg, 0.065 mmol) following thesame procedure described above to give 32 mg (57%) as a clear film afterflash chromatography (20-100% EtOAc/Hex)

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.44 (d, J=8.5 Hz, 1.3H), 7.32(d, J=8.5 Hz, 0.7H), 6.95-6.77 (m, 2H), 6.62 (dd, J=9.2, 1.7 Hz, 1H),6.09 (d, =9.1 Hz, 1H), 5.24 (s, 0.7H), 5.13-4.95 (m, 1H), 4.84 (s,0.3H), 4.69 (d, J=9.6 Hz, 0.3H), 4.60 (d, J=9.0 Hz, 0.7H), 4.19 (q,J=7.1 Hz, 2H), 4.09 (td, J=4.7, 2.4 Hz, 2H), 3.84 (t, J=4.9 Hz, 2H),3.72 (dd, J=5.7, 3.2 Hz, 2H), 3.70-3.65 (m, 2H), 3.66-3.62 (m, 4H), 3.60(t, J=6.5 Hz, 2H), 3.09 (t, J=6.5 Hz, 2H), 2.96-2.88 (m, 3H), 2.84 (s,3H), 2.33 (s, 3H), 1.88 (d, J=3.5 Hz, 3H), 1.49 (s, 2H), 1.43 (d, J=5.5Hz, 11H), 1.35 (s, 2H), 1.30 (t, J=7.1 Hz, 2H), 0.87 (d, j=6.6 Hz, 3H),0.80 (d, J=15.9 Hz, 3H), 0.76 (s, 9H). C₄₄H₇₃N₃O₁₁S calcd. [M+H]⁺=852.51amu. found m/z=852.79. R_(f)=0.60 (60% EtOAc/Hex).

Example 138

(6S,9S,12S,E)-ethyl9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(3-((16-oxo-3,6,9-trioxa-12-thiatetradecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate

The title compound was prepared from(S)-3-(3-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methyl-2-(methylamino)butanoicacid (73 mg, 0.080 mmol) following the same procedure described above togive 66 mg (47%) as a clear film after flash chromatography (20-100%EtOAc/Hex).

¹H NMR (400 MHz, Chloroform-d) δ (ppm) 7.25-6.92 (m, 3H), 6.78-6.70 (m,1H), 6.62 (d, J=8.9 Hz, 1H), 6.12 (d, J=8.9 Hz, 1H), 5.26 (s, 0.7H),5.12-4.99 (m, 1H), 4.89 (s, 0.3H), 4.74-4.56 (m, 1H), 4.19 (q, J=7.2 Hz,1H), 4.16-4.03 (m, 2H), 3.84 (td, J=5.0, 3.2 Hz, 2H), 3.77-3.61 (m,14H), 3.60 (t, J=6.4 Hz, 2H), 3.09 (t, J=6.5 Hz, 2H), 2.97-2.75 (m, 6H),2.33 (s, 3H), 1.91-1.83 (m, 3H), 1.52-1.35 (m, 16H), 1.26 (t, J=7.1 Hz,3H), 0.87 (d, J=6.0 Hz, 3H), 0.81 (d, J=12.9 Hz, 3H), 0.77 (s, 9H).C₄₆H₇₇N₃O₁₂S calcd. [M+H]⁺ =896.53 amu. found m/z=896.68. R_(f)=0.61(75% EtOAc/Hex).

Example 139

(S,E)-4-((S)-2-((S)-3-(4-((14-mercapto-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoicacid disulfide

The title compound was prepared by saponification, then TFA promoted Bocremoval, according to the exact methods described in Nieman et al. from(6S,9S,2S,E)-ethyl9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4-((16-oxo-3,6,9,12-tetraoxa-15-thiaheptadecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate(26 mg, 0.029 mmol) to afford the title compound (16 mg, 90%) as a clearglass after complete removal of excess TFA.

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 8.43 (d, J=8.1 Hz, 1H), 7.47 (d,J=8.5 Hz, 2H), 7.08-6.94 (m, 2H), 6.80 (dq, J=9.9, 1.5 Hz, 1H), 5.08 (t,J=10.1 Hz, 1H), 4.94 (d, J=8.1 Hz, 1H), 4.32 (s, 1H), 4.21-4.12 (m, 2H),3.93-3.81 (m, 3H), 3.76 (t, J=6.4 Hz, 2H), 3.76-3.72 (m, 2H), 3.72-3.62(m, 10H), 3.17 (s, 3H), 2.92 (t, J=6.4 Hz, 2H), 2.61-2.47 (m, 3H),2.14-2.00 (m, 1H), 1.94 (d, J=1.5 Hz, 3H), 1.46 (s, 3H), 1.40 (d, J=7.7Hz, 3H), 1.09 (s, 9H), 0.94 (d, J=5.0 Hz, 3H), 0.92 (d, J=4.8 Hz, 3H).C₇₄H₁₂₄N₆O₁₈S₂ calcd. [M+H]⁺ =1449.85 amu. found m/z=1450.49.

Example 140

Compound of Example 139 is reduced according to the methods below toproduce the subject compound.

Example 141

(S,E)-4-((S)-2-((S)-3-(4-(2-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)ethoxy)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoicacid disulfide

The title compound was prepared by saponification, then TFA promoted Bocremoval, according to the exact methods described in Nieman et al. from(6S,9S,12S,E)-ethyl9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(4-((13-oxo-3,6,9-trioxa-12-thiatetradecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate(32 mg, 0.037 mmol) to afford the title compound (29 mg, 86%) as a clearglass after complete removal of excess TFA.

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 8.39 (d, J=8.2 Hz, 1H), 7.44 (d,J=8.9 Hz, 2H), 7.01 (d, J=8.5 Hz, 2H), 6.77 (d, J=7.9 Hz, 1H), 5.05 (t,J=10.1 Hz, 1H), 4.92 (d, J=8.3 Hz, 1H), 4.28 (s, 1H), 4.15 (dd, J=5.8,3.4 Hz, 2H), 3.89-3.80 (m, 2H), 3.73 (t, =6.4 Hz, 2H), 3.72-3.69 (m,2H), 3.69-3.60 (m, 6H), 3.14 (s, 3H), 2.89 (t, J=6.4 Hz, 2H), 2.50 (s,3H), 2.11-1.97 (m, 1H), 1.91 (d, J=1.4 Hz, 3H), 1.43 (s, 3H), 1.36 (s,3H), 1.06 (s, 9H), 0.92-0.87 (m, 6H). C₇₀H₁₁₈N₆O₁₆S₂ calcd.[M+H]⁺=1361.80 amu. found m/z=1362.26.

Example 142

Compound of Example 141 is reduced according to the methods below toproduce the subject compound.

Example 143

(S,E)-4-((S)-2-((S)-3-(3-((14-mercapto-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoicacid

The title compound was prepared by saponification, then TFA promoted Bocremoval, according to the exact methods described in Nieman et al. from(6S,9S,12S,E)-ethyl9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-6-(2-(3-((16-oxo-3,6,9,12-tetraoxa-15-thiaheptadecyl)oxy)phenyl)propan-2-yl)-3-oxa-5,8,11-triazapentadec-13-en-15-oate(56 mg, 0.029 mmol) to afford the title compound (43 mg, 82%) as anoff-white foam after complete removal of excess TFA.

¹H NMR (400 MHz, Methanol-d₄) δ (ppm) 8.48 (d, J=8.3 Hz, 1H), 7.47-7.29(m, 1H), 7.21-7.04 (m, 1H), 6.95 (t, J=9.4 Hz, 1H), 6.80 (d, J=9.7 Hz,1H), 5.08 (t, J=10.1 Hz, 1H), 4.97-4.94 (m, 1H), 4.38 (s, 1H), 4.24-4.13(m, 2H), 3.95-3.82 (m, 2H), 3.80-3.58 (m, 14H), 3.17 (s, 3H), 2.92 (t,J=6.4 Hz, 2H), 2.53 (s, 3H), 2.11-2.03 (m, 1H), 1.94 (d, J=1.4 Hz, 3H),1.47 (s, 3H), 1.40 (s, 3H), 1.09 (s, 9H), 0.93 (dt, J=11.2, 3.4 Hz,15H). C₇₄H₁₂₄N₆O₁₈S₂ calcd. [M+H]⁺=1449.85 amu. found m/z=1450.06.

Example 144

Compound of Example 143 is reduced according to the methods below toproduce the subject compound.

Example 145

(mAb—SPDP—Compound 142) produced using the Compound 142 synthesismethod, above, and the SPDP linkage method described below.

Example 146

(mAb—SPDP—Compound 140) produced using the Compound 140 synthesismethod, above, and the SPDP linkage method described below.

Example 147

(mAb—SPDP—Compound 144) produced using the Compound 144 synthesismethod, above, and the SPDP linkage method described below.

Example 148

(mAb—SMCC—Compound 140) produced using the Compound 140 synthesismethod, above, and the SMCC linkage method described below.

Example 149

(mAb—SMCC—Compound 142) produced using the Compound 142 synthesismethod, above, and the SMCC linkage method described below.

Example 150

(mAb—SMCC—Compound 144) produced using the Compound 144 synthesismethod, above, and the SMCC linkage method described below.

Other Examples Example 151

(S,E)-N-(benzylsulfonyl)-4-((S)-2-((S)-3-cyclohexyl-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enamide

The title compound was synthesized from(S)-2-(tert-butoxycarbonyl(methyl)amino)-3-cyclohexyl-3-methylbutanoicacid as prepared by Zask et al., J. Med. Chem. 2004, 47, (19), 4774-4786and(S,E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide,prepared using General Procedures 10, 11, 3 and 2 by application ofGeneral Procedures 4 and 7.

¹H NMR (400 MHz, Methanol-d₄) δ 7.38 (s, 5H), 6.37 (dd, J=9.4, 1.7 Hz,1H), 5.01 (t, J=10.0 Hz, 1H), 4.91 (s, 1H), 4.75 (s, 2H), 4.01 (s, 1H),3.10 (s, 3H), 2.66 (s, 3H), 2.05-1.91 (m, 4H), 1.91-1.67 (m, 6H),1.45-1.28 (m, 3H), 1.29-1.01 (m, 17H), 0.95-0.75 (m, 9H).

C34H56N4O5S calcd m/z=632.40. found [M+H]⁺=633.35.

Example 152

(mAb—MCvcPABC—Compound 85) produced using Example compound 120, above,and the general MCvcPABC conjugation method described below.

Example 153

(mAb—MCvcPABC—Compound 77) produced using Example compound 119, above,and the general MCvcPABC conjugation method described below.

Example 154

(mAb—MCvcPABC—Compound 80) produced using Example compound 121, above,and the MCvcPABC conjugation method described below.

Example 155

(mAb—MCvcPABC—Compound 58) produced using Example compound 158(MCvcPABC58), below, and the MCvcPABC conjugation method describedbelow.

Example 156

(mAb—MCvcPABC—Compound 41) produced using Example compound 122, above,and the MCvcPABC conjugation method described below.

Example 157

(mAb—MCvcPABC—Compound 63) produced using Example compound 159(MCvcPABC830), below, and the MCvcPABC conjugation method describedbelow.

Example 158

The title compound was prepared by application of General Procedure 15and 7 to Boc protected Example 58.

¹H NMR (400 MHz, Methanol-d₄) δ 7.60 (d, J=8.1 Hz, 2H), 7.56 (d, =7.8Hz, 2H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (t, J=7.3 Hz, 1H), 7.33 (d, J=8.2Hz, 2H), 7.26 (d, J=8.0 Hz, 2H), 7.22 (d, =7.9 Hz, 2H), 6.81 (s, 2H),6.37 (d, J=9.3 Hz, 1H), 5.13-5.01 (m, 3H), 4.96 (s, 1H), 4.70 (s, 2H),4.56-4.51 (m, 1H), 4.38 (s, 1H), 4.23-4.16 (m, 1H), 3.50 (t, J=7.1 Hz,2H), 3.27-3.19 (m, 1H), 3.18-3.04 (m, 4H), 2.52 (s, 3H), 2.30 (t, =7.4Hz, 2H), 2.15-2.05 (m, 1H), 1.96 (s, 3H), 1.98-1.88 (m, 1H), 1.83-1.73(m, 1H), 1.64 (dq, J=23.1, 7.3 Hz, 7H), 1.48 (s, 3H), 1.39 (s, 3H),1.37-1.30 (m, 2H), 1.27 (s, 2H), 1.21 (s, 2H), 1.08 (s, 9H), 1.00 (d,J=6.7 Hz, 3H), 0.99 (d, J=6.8 Hz, 3H), 0.91 (d, J=6.6 Hz, 3H), 0.88 (d,J=6.5 Hz, 3H).

C₆₆H₉₃N₁₁O₁₃S calcd. m/z=1279.7. found [M+H]⁺ =1281.0.

Example 159

The title compound was prepared by application of General Procedures 15and 7 to Boc protected Example 63.

C₆₅H₉₁N₁₁O₁₃S calcd. m/z=1265.7. found [M+H]⁺ =1266.7.

It is understood to those skilled in the art that it may be possible tocarry out the chemical conversions shown in the schemes above withmodifications of one or more parameters. As examples, alternatenon-nucleophilic solvents may be suitable for the chemistry, such asTHF, DMF, Toluene etc. Reaction temperatures may be varied. Alternatereagents may be suitable to act as dehydrating or acid-activating agentswhich are normally used in amide formation reactions, such aspentafluorophenyl esters, NHS esters, EDAC, HBTU, HOBT etc.

Other Representative Compounds

The following representative compounds may be prepared according to theforegoing procedures. As recognized by the artisan of reasonable skill,the following compounds are synthetically accessible using thedisclosure of WO 2004/026293 to achieve the precursor reactant andapplying General Procedures with the appropriate sulfonamide.

Example 1 Biological Assays

Tables 1-8 summarize the cytotoxic activity of the subject compounds oncell lines. FIG. 1 summarizes the data for compounds A, B, C, D, and Ewhen tested using the Human mammary carcinoma cell line HCC1954 or HumanT-cell leukemia cell line Jurkat. FIGS. 2-6 show the cytotoxicity dataplots for individual compounds A-E. Tables 2-6 summarize the results ofadditional cytotoxicity assays.

Cell lines used: Human T-cell leukemia cell line Jurkat (ATCC: TIB-152);HCC1954 (ATCC: CRL. 2338); Human Pancreatic cells lines: AsPC-1 (ATCC:CRL-1682), BxPC-3 (ATCC: CRL.1687), HPAF-II (ATCC: CRL.1997), MiaPaCa2(ATCC: CRL.1420), PANC-1 (ATCC: CRL.1469), Capan-1 (ATCC: HTB-79),Capan-2 (ATCC: HTB-80) and the Human gastric carcinoma cell line NCI-N87(ATCC: CRL. 5822); AML-193 (ATCC: CRL.9589), CCRF-CEM (ATCC: CCL-119),DU145 (ATCC: HTB-81), PC-3 (ATCC: CRL.1435), A-431 (ATCC: CRL.1555),HT-29 (ATCC: HTB-38), A-172 (ATCC: CRL.1620), NCI-H358 (ATCC: CRL.5807),A549 (ATCC: CCL-185), Colo-205 (ATCC: CCL-222), MDA-MB-231 (ATCC:HTB-26), OVCAR-3 (ATCC: HTB-161), OV-90 (ATCC: CRL.11732), OE19 (Sigma:96071721), RT112/84 (Sigma: 85061106).

On the day prior to adding compounds, HCC1954 AsPC-1, BxPC-3, HPAF-11,MiaPaCa2, PANC-1, Capan-1, Capan-2 and NCI-N87 cells were added toopaque-walled 96-well tissue culture-treated microtiter plates usingcomplete growth medium at a density of 2500 cells/100 microliter (uL) ofmedium. These adherant cell lines cells were incubated for one night at37° C./5% CO₂ to allow the cells to attach to the microtiter platesurface. On the day that compounds were added, Jurkat cells are added toseparate 96-well microtiter plates at 2500 cells/100 uL using the samegrowth medium as HCC1954. Compound were first serially diluted usingdimethyl sulfoxide, and then the prepared dilutions are added tocomplete growth medium at five-times the final concentration—compoundswere then titrated 1:3, eight steps. A control with no compound (growthmedium alone) was included on each microtiter plate in sextuplicate. Theprepared compounds titrations were added (twenty-five uL/well) intriplicate. The cells and compound titrations were incubated at 37°C./5% CO₂ for three nights. After the incubation, cell viability ismeasured using CellTiter-Glo® reagent by adding thirty uL of preparedCellTiter-Glo® to each assay well. The assay is incubated for at leasttwenty minutes in the dark prior to measuring emitted luminescence usinga microplate luminometer (500 ms integration time). The collectedrelative luminescence units (RLU) are converted to % cytotoxicity usingthe Growth medium alone control mentioned above (% Cytotoxicity=1−[WellRLU/average medium alone control RLU]).

GraphPad Prism was used for generation of EC₅₀ values using threeparameter non-linear regression curve fitting.

TABLE 1 Cytotoxicity of Compounds HCC1954 cells (HER2+) Jurkat cells(HER2−) COM- EC₅₀ EC₅₀ bounds EC₅₀ EC₅₀ bounds POUND (nM) (nM) (nM) (nM)A 0.86 0.3765 to 1.966 0.78 0.5970 to 1.013  B 8.1  4.778 to 13.56 10.56.221 to 17.70 C 0.67 0.3738 to 1.186 0.57 0.4088 to 0.8085 D 0.061 0.04550 to 0.08050 0.043  0.03127 to 0.05921 E 0.79 0.5418 to 1.1401.67 1.223 to 2.268

TABLE 2 Cytotoxicity of Compounds HCC1954 Jurkat EC₅₀ (nM) EC₅₀ bounds(nM) R square EC₅₀ (nM) EC₅₀ bounds R square A 3 1.582 to 5.228 0.9158 53.127 to 6.641 0.9647 B 13 10.50 to 16.27 0.9878 59 33.41 to 104.50.9257 C 1.3 0.7970 to 1.977  0.9493 1.9 1.248 to 2.896 0.9562 D 0.060.04550 to 0.08050 0.9656 0.04 0.03127 to 0.05921 0.9497 E 0.79 0.5418to 1.140  0.9314 1.67 1.223 to 2.268 0.9518

TABLE 3 Cytotoxicity of Compounds on Jurkat Cells Compound EC₅₀ (nM) A4.5 B 59 C 1.9 118  13 D 0.033 E 1.67 12 0.030 13 0.038 14 0.007 140.015 15 7.604 16 0.041 17 0.325 18 1.358 19 0.152 22 0.021 47 0.261 240.070 48 0.208 23 0.031 28 0.021 29 0.121 30 0.109 31 0.094 74 0.087 250.050 26 0.105 49 2.5 50 0.171 27 0.157 32 0.265 76 0.328 79 0.386 841.393 80 0.389 51 0.247 57 0.566 58 0.816 34 0.200 97 1.616 44 0.114 450.869 42 0.165

TABLE 4 Cytotoxicity of Compounds on HCC-1954 Cells Compound EC₅₀ (nM) A2.1 B 13 C 1.3 D 0.06 E 0.79 79 0.241 80 0.207

TABLE 5 Cytotoxicity (EC₅₀) of Compounds on Various Tumour Cell Lines(nM) NCI- HPAF- PANC- Capan- Compound N87 AsPC-1 BxPC-3 II MiaPaCa2 1Capan-1 2 D 0.272 0.1704 0.06635 0.177 0.136 0.806 — — 14 0.175 0.2060.0458 0.172 0.204 1.356 2.081 1.103 24 — 0.5857 0.2704 0.396 0.5662.181 — — 23 0.402 — — — — — — — 77 — 15.53 36.5 17.240 94.290 97.190 —— 63 — 0.9697 0.6973 0.826 1.018 3.997 — —

TABLE 6 Compound Cytotoxicity on Jurkat Compound EC₅₀ (nM) 108 0.017 1100.031 107 0.043 114 0.056 112 0.064 98 0.077 109 0.087 91 0.109 64 0.13866 0.145 93 0.196 103 0.209 104 0.272 95 0.288 102 0.289 97 0.307 680.337 45 0.373 92 0.485 72 0.531 67 0.562 33 0.636 88 0.641 105 0.731105 0.753 35 0.832 70 0.856 71 1.021 62 1.195 44 1.479 13 1.515 69 1.56494 1.673 73 2.684 96 10.260 111 ~0.1178 91 0.109 93 0.196 95 0.288 970.307 92 0.485 88 0.641 62 1.195 94 1.673 96 10.260 64 0.138 66 0.145103 0.209 104 0.272 102 0.289 68 0.337 72 0.531 105 0.731 105 0.753 700.856 71 1.021 69 1.564 46 — 108 0.017 110 0.031 107 0.043 114 0.056 1120.064 98 0.077 109 0.087 111 0.12 97 0.307 45 0.373 44 1.479 67 0.562 330.636 35 0.832 72 2.684

TABLE 7 Cytotoxicity on Jurkat Compound EC₅₀ (nM) 107 0.043 108 0.017109 0.087 110 0.031 111 0.12 112 0.064 114 0.056

TABLE 8 Cytotoxicity on Various Cell Lines Compound-14 (EC₅₀) TumourCell Line (nM) AML-193 0.191 CCRF-CEM 0.130 DU145 0.649 PC-3 0.455 A-4310.191 HT-29 0.167 HCC-1954 0.131 A-172 0.598 NCI-N87 0.325 Jurkat 0.068BxPC-3 0.196 NCI-H358 0.311 Mia PaCa-2 0.332 A549 0.860 Colo-205 ~0.3168PANC-1 0.759 MDA-MB-231 1.242 AsPC-1 0.334 HPAF-II ~0.3850 OVCAR-3 0.090OV-90 0.515 OE19 0.210 RT112/84 0.178

Example 2 Exemplary Antibody-Drug Conjugates

Antibody-Drug Conjugates—Exemplary Linkers

As recognized by the artisan of reasonable skill, the particular linkerused for conjugate formation will depend upon the reactive group of thereactant compound being used for bond formation. As an example, andwithin the scope of the present invention, compounds having thiol moietymay be used for conjugate formation. In some of the present examples,the commercially available cleavable linker sulfosuccinimidyl6-[3′(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP: ThermoPierce Cat#21650) and Non-cleavable linker succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC: Thermo PierceCat#22360) were utilized for antibody-drug conjugation reactions. Thecoupling procedure is performed in two major steps: 1) incorporation ofthe linkers onto the antibody via reaction with antibody primary aminegroups (Lysine residues) and the N-hydroxysuccinimide (NHS) ester moietyof the linkers, and 2) reaction of the incorporated maleimide group(SMCC) or 2-pyridyldithio group (LC-SPDP) with thiol-containingcompounds.

Activation of Antibody with Cleavable (LC-SPDP) or Non-Cleavable (SMCC)Linkers

Antibody (Herceptin) was diluted into either Potassium Phosphate pH 8(sulfo-LC-SPDP) or D-PBS (Invitrogen) pH 7.4 (SMCC) to 5 mg/mL. To thediluted antibody, freshly dissolved linker was added—using ultra-purewater for sulfo-LC-SPDP or anhydrous N,N-Dimethylacetamide (DMA) forSMCC. 10-14 fold molar-excesses of SMCC:antibody orsulfo-LC-SPDP:antibody result in incorporation of 5-7 linkers/antibody.The linker-antibody “activation” reaction was incubated at 28° C. for 2hours. Following the incubation, the un-reacted linker was removed fromeach antibody sample using 40 kda Zeba Size-exclusionchromatography/desalting columns (Thermo Pierce Cat#87771, or 87772depending on the scale). During the same chromatography step the bufferwas exchanged in preparation for the next reaction; either PhosphateBuffer/EDTA pH 6.5 (LC-SPDP), or Citrate buffer/EDTA pH 5 (SMCC). Thepurified preparations were then assayed for total protein content versusan antibody standard curve using the microplate adapted BCA assay(Thermo Pierce Cat#23225). To estimate the extent of linkerincorporation a small scale reaction with excess (˜10-fold compared toprotein concentration) Cysteine was performed. Following a 10 minuteincubation the un-reacted Cysteine was detected using5,5-Dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent, Thermo PierceCat#22582). By interpolating the concentration from a Cysteine standardcurve the linker concentration was determined by subtracting thedetermined value from the known concentration of Cysteine used.

Reaction of Thiol-Containing Compounds to Linker-Activated Antibody

In the second step of the coupling reaction, the activated-antibody wasutilized by first diluting the preparation to 2 mg/mL using eitherPhosphate Buffer/EDTA pH 6.5 (LC-SPDP), or Citrate buffer/EDTA pH 5(SMCC). Prior to use, the thiol containing n-acyl sulfonamide compoundsor maytansinoid DM1 were reduced using TCEP-agarose beads to ensure thethiol group was available to react to the incorporated linkers. Inbrief, compounds were diluted to 5 mM using Phosphate Buffer/EDTA pH6.5. In instances where aqueous solubility was an issue, a small volumeof 37% HCl (1:300) was added and this was sufficient to solubilize thecompounds at 5 mM. TCEP-agarose beads (Thermo Pierce Cat 77712), wereequilibrated with Phosphate Buffer/EDTA/10% DMA prior to use. Thecompound dilutions were rotated with TCEP-agarose beads for at least 0.5hours, or up to 3 hours. The reduced compounds were collected bycentrifugation over a filter which excluded the TCEP-agarose. The extentof reduction and thiol concentration was measured using Ellman's reagent(compared to a Cysteine standard curve). The reduced thiol-containingcompounds were then added to the activated antibody samples at a molarexcess of ˜2-fold compared to the previously determined linkerconcentrations. In order to monitor the coupling reaction effectivenessan “overnight” conjugation control was prepared by diluting eachcompound into Phosphate Buffer/EDTA pH 6.5 or Citrate buffer/EDTA pH 5at the same dilution factor that was used in the conjugation reaction.The remaining compound stocks were frozen at −80° C. The reactions andovernight controls were incubated at ambient temperature overnight. Thenext morning the frozen compound stocks were thawed and another controlwas prepared for each compound exactly like the “overnight” control—thisis the “fresh” control. A small volume of each conjugation reaction wascompared to the overnight and fresh compound controls using Ellman'sreagent. Non-reacted compound was purified away from the ADCs using 40kda Zeba Size-exclusion/desalting columns; during the same step thebuffer was exchanged to D-PBS pH7.4 (Invitrogen).

The purified ADCs were then analysed for: total protein content (BCAassay, Pierce microBCA protocol), relative affinity for antigen binding(equilibrium native binding), and selective cytotoxic killing ofHER2-positive cells (HCC1954) compared HER2-negative cells (Jurkat).

Cytotoxicity Assay

Tables 9 and 10 summarize the cytotoxic activity of ADCs comprisingcompounds A, B, or C when tested using the Human mammary carcinoma cellline HCC1954 or Human T-cell leukemia cell line Jurkat. FIGS. 7-9 showcytotoxicity data plots for individual compositions as indicated.

On the day prior to adding test articles, HCC1954 cells were added toopaque-walled 96-well tissue culture-treated microtiter plates usingcomplete growth medium at a density of 2500 cells/100 microliter (uL) ofmedium. The HCC1954 cells were incubated for one night at 37° C./5% CO₂to allow the cells to attach to the microtiter plate surface. On the daythat test articles were added, Jurkat cells are added to separate96-well microtiter plates at 2500 cells/100 uL using the same growthmedium as HCC1954. To compare the ADC killing to that obtained from thefree-compounds, the n-acyl sulfonamide compounds were first seriallydiluted using dimethyl sulfoxide or DMA, and then the prepared dilutionsare added to complete growth medium at five-times the finalconcentration—compounds were then titrated 1:3, eight steps. To test theADCs, they were diluted directly in growth medium at five-times thefinal concentration—ADCs were then titrated 1:3, eight steps. A controlwith no test article present (growth medium alone) was included on eachmicrotiter plate in sextuplicate. The prepared compound/ADC titrationswere added (twenty-five uL/well) in triplicate to both the HCC1954 cellsand Jurkat cells. The cells and titrations were incubated at 37° C./5%CO₂ for three nights. After the incubation, cell viability was measuredusing CellTiter-Glo® reagent by adding thirty uL of preparedCellTiter-Glo® to each assay well. The assay was incubated for at leasttwenty minutes in the dark prior to measuring emitted luminescence usinga microplate luminometer (500 ms integration time). The collectedrelative luminescence units (RLU) were converted to % cytotoxicity usingthe Growth medium alone control mentioned above (% Cytotoxicity=1−[WellRLU/average medium alone control RLU]).

The data indicate that the subject compounds are active cytotoxins onboth cell lines used. The LC-SPDP-linked compound conjugatesdemonstrated potent killing of HER2-positive HCC1954 cells. Jurkat cellkilling was observed at high-doses of ADC due to the presence ofβ-mercaptoethanol in cell culture medium, which resulted in the releaseof free compound (data not shown).

TABLE 9 Cytotoxicity - Coupling #1 HCC1954 Jurkat Best-fit Best-fit EC₅₀Bounds EC₅₀ Bounds (nM) EC₅₀ (nM) (nM) EC₅₀ (nM) SMCC-Herceptin-SMCC-Compound A 6.5 2.740 to 15.22 332 134.6 to 819.0 linkedHerceptin-SMCC-Compound B 66 26.48 to 165.1 83 48.29 to 144.0Herceptin-SMCC-Compound C 6 2.966 to 12.79 12 6.594 to 20.26 LC-SPDP-Herceptin-LC-SPDP- 0.86 0.6660 to 1.121  21 13.74 to 32.68 linkedCompound A Herceptin-LC-SPDP- 0.068 0.02234 to 0.2093  11 7.028 to 15.91Compound B Herceptin-LC-SPDP- 0.070 0.02590 to 0.1914  2 1.521 to 3.613Compound C Free Compound A 2.1 1.352 to 3.280 1.1 0.7580 to 1.473 Compounds Compound B 8.1 4.778 to 13.56 10 6.221 to 17.70 Compound C — —— —

TABLE 10 Cytotoxicity-Coupling #2 HCC1954 Jurkat Best-fit BoundsBest-fit Bounds EC₅₀ (nM) EC₅₀ (nM) EC₅₀ (nM) EC₅₀ (nM) SMCC-linkedHerceptin-SMCC- 15 8.266 to 27.50 50 28.62 to 87.34 Compound A not doneHerceptin-SMCC- Compound B Herceptin-SMCC- Compound C LC-SPDP-Herceptin-LC-SPDP- 0.061 0.01410 to 0.2672  8.7 5.852 to 12.96 linkedCompound A Herceptin-LC-SPDP- 0.22 0.1381 to 0.3441 14 9.469 to 21.41Compound B Herceptin-LC-SPDP- 0.042 0.01371 to 0.1275  1.6 1.160 to2.110 Compound C Free Compound A 0.86 0.3765 to 1.966  0.78 0.5970 to1.013  Compounds Compound B 9.2 5.300 to 15.98 36 20.52 to 64.36Compound C 0.67 0.3738 to 1.186  0.57 0.4088 to 0.8085

Analysis of Antibody-Drug Conjugate (ADC) by EsiToF Mass Spectrometry.

Electrospary ionization time of flight (EsiToF) mass spectrometerinstrument—QStar XL Hybrid quadrupole-TOF LC/MSMS—(AB Sciex) was used todetermine molecular weight of the ADC's and to evaluate thedrug-to-antibody ratio (DAR). The EsiToF MS instrument was equipped withelectrospray ionization turbo spray source. Data acquisition wasperformed in the positive ion mode, and the sample's total ion currentwas acquired over the mass range 2000 m/z to 4000 m/z using Analyst QS1.1 software. The ion source was operated with an ion spray needlevoltage of 5.2 KV, and a nebulization (Gas 1) at 25 (arbitrary units),curtain gas of 30 (arbitrary units), declustering potential of 150 V andat temperature of 150° C. The. The ADC test sample solutions wasintroduced at 5 uL/min into the ion source by direct infusion via afused silica capillary with the help of syringe and syringe pump.

Preparation of the ADC Sample for ESI-ToF MS Analysis

All ADC sample were deglycosylated using EndoS(IgGZERO)™ endoglycosidaseand buffer exchanged with water prior to EsiToF-MS analysis. Briefly,the original ADC sample was run through a 100K MWCO Amicon concentratorfor buffer exchange in sodium phosphate buffer. The buffer exchangedsample was then treated with IgGZERO (1 unit/1 ug of antibody) in sodiumphosphate cleavage buffer, containing 150 mM NaCl, and incubated for 30minutes at 37° C. The resulting deglycosylated ADC was again bufferexchanged with water using a 100K MWCO Amicon concentrator, and dilutedwith 0.1% formic acid in acetonitrile/water (50/50 v/v %) to aconcentration of 3.0 μg/μL prior to analysis.

Analyses indicated that antibody was loaded with a DAR range of between4-7 (data not shown).

Example 3 Exemplary Antibody-Drug Conjugates

Preparation of Antibody-Drug Conjugates from MCvcPABC-Toxins, GeneralMethods:

To a solution of antibody (1-10 mg/mL) in 25 mM sodium borate, 25 mMsodium chloride, 1 mM DTPA (pH 8.0) was added TCEP from a freshlyprepared stock (1-10 mM) in the same buffer (2.0-3.0 molar equivalents).The solution was mixed thoroughly and incubated at 37° C. for two hoursbefore cooling on ice. In some instances the reduced antibody solutionwas further diluted with either ice-cold phosphate buffered salinecontaining 1 mM DTPA (final protein concentration 2.0 mg/mL) or ice-cold25 mM sodium borate, 25 mM sodium chloride, 1 mM DTPA (pH 8.0), toobtain a solution with a final protein concentration of between 1 and 4mg/mL. To the reduced protein solution stored on ice was added themaleimide functionalized toxin (10-12 molar equivalents) from a 10 mMdmso stock solution. The conjugation reaction was immediately mixedthoroughly by inversion and conjugation was allowed to proceed on icefor a period of approximately 1 hour before purification by passage overZeba Spin Desalting Columns (40 KDa MWCO; Peirce) pre-equilibrated withphosphate buffered saline or 10 mM sodium citrate, 150 mM sodiumchloride, pH 5.5. The eluate was pooled, filter sterilized (Steriflip,Millipore), and stored at 4° C.

The purified ADCs were analyzed for total protein content (bicinchonicacid assay, Pierce microBCA protocol, catalogue #23225). The ADC productwas characterized by reducing and non-reducing PAGE, HPLC-HIC, SEC, andRP-UPLC-MS. The average DAR and drug distribution were derived frominterpretation of HIC and LC-MS data with reference to non-reducingPAGE. Average DAR estimates were normally in the range of 3.5-4.5.Relative affinity of ADCs for antigen binding (equilibrium nativebinding) was performed as described (above/below). The selectivecytotoxicity of the antibody drug conjugates was assessed by testing forkilling of both antigen positive and antigen negative cell lines.

Assay of Selective In Vitro Cytotoxicity of Antigen-Positive Cells byAntibody Drug Conjugates:

Selective killing of an antigen positive cell line (including HCC1954,NCI-N87, HPAF-II and BxPC-3 cell lines) over antigen-negative Jurkatcells was demonstrated for each conjugate prepared. Cytotoxicity ofexample ADCs on several antigen positive cell lines is summarized in theidentified Figures and Tables 9-13. In addition, the conjugatesindicated by (*) in Table 11 were tested and showed potent cell killactivity against a human breast cancer cell line (data not shown).Briefly, cells were obtained from the ATCC and cultured as described inthe product sheet provided. Cells were seeded at 25000 cells/mL (2500cells/well) in Costar 3904 black walled, flat bottomed 96-well plates.Adherent cell lines cells were incubated for one night at 37° C. in a 5%CO₂ atmosphere to allow the cells to attach to the microtitre platesurface, while suspension (Jurkat) cells were plated immediately beforeuse. ADCs were diluted directly in the appropriate cell growth medium atfive-times the desired final concentration. These ADCs were thentitrated, normally 1:3, over eight steps. A control with no test articlepresent (growth medium alone) was included on each microtiter plate insextuplicate. The prepared ADC titrations were added (25 uL/well) intriplicate to each cell line assayed. The cells and titrations wereincubated at 37° C./5% CO₂ for three nights (Jurkat) and five nights(all other cell lines). After the incubation, cell viability wasmeasured using CellTiter-Glo® reagent by adding thirty uL of preparedCellTiter-Glo® to each assay well. The mixtures were incubated for atleast twenty minutes in the dark prior to measuring emitted luminescenceusing a microplate luminometer (500 ms integration time). The collectedrelative luminescence units (RLU) were converted to % cytotoxicity usingthe growth medium alone control mentioned above (% Cytotoxicity=1−[WellRLU/average medium alone control RLU]). Data (% Cytotoxicity vs.Concentration of ADC (log 10(nM)) were plotted and were analyzed bynon-linear regression methods using GraphPad Prism software v. 5.02 toobtain EC₅₀ estimates.

Estimation of Drug to Antibody Ratio (Dar):

The average degree of conjugation of toxin-linker to antibody wasassessed by hydrophobic interaction chromatography and high performanceliquid chromatography-mass spectrometry. These techniques are describedin Antibody Drug Conjugates, Methods in Molecular Biology vol. 1045,2013. pp 275-284. L. Ducry, Ed., and Asish B. Chakraborty, Scott J.Berger and John C. Gebler, Characterization of an IgG1 MonoclonalAntibody and related Sub-structures by LC/ESI-TOF/MS: Application note,Waters Corporation. March 2007. 720002107EN.

Method 1. Hydrophobic Interaction Chromatography

Antibody drug conjugates were subjected to hydrophobic interactionchromatography (HIC) on a TSKgeI Butyl-NPR column (Tosoh Bioscience; 4.6mm×35 mm i.d.; 2.5 μm particle size) connected to an Agilent 1100 seriesHPLC. Samples were injected (5 uL) at or above 4 mg/mL. Where necessary,ADCs were concentrated prior to injection using PALL Nanosep Omegacentrifugal concentration devices (part # OD010C34). A linear gradientelution was employed starting at 95% mobile phase A/5% mobile phase B,transitioning to 5% mobile phase A/95% mobile phase B over a period of12 minutes (mobile phase A: 1.5M ammonium sulfate+25 mM sodium phosphateat pH 6.95 and mobile phase B: 25% isopropanol, 75% 25 mM sodiumphosphate at pH 6.95). Injection of unmodified antibody provided a meansof identifying the peak with DAR=0. Antibodies were detected on thebasis of absorbance at 280 nm.

Method 2. Ultra Performance Liquid Chromatography-Mass Spectrometry forDAR Estimation

Reversed phase ultra performance liquid-chromatography tandemESI-QToF-mass spectrometry (UPLC-ESI-QToF-MS) was used to characterizeantibody drug conjugates for extent of drug conjugation followingreduction with dithiothreitol. The characterization was performed usingAcquity-UPLC (H-class) Bio coupled to a Quatro-Premier QToF massspectrometer with an electrospray ion source (WATERS Corporation). UPLCanalysis of the reduced ADC sample was performed at 70° C. with aPolymerX 5u PR-1 100 A, 50×2.0 mm column (Phenomenex, Inc.) and with amobile phase composed of Solvent A: Acetonitrile/Water/Trifluoroaceticacid/Formic acid (10/90/0.1/0.1, v/v %), and Solvent B:Acetonitrile/Formic acid (100/0.1, % v/v). Components of the reduced ADCsample were eluted with a linear gradient starting at Solvent A/SolventB (80/20 v/v and a flow rate of 0.3 ml/min to Solvent A/Solvent B(40/60, v/v %) over 25 min, and then to Solvent A/Solvent B (10/90, v/v%) over 2 minutes before equilibrating back to initial conditions. Thetotal run time was 30 minutes. The ESI-Tof MS total ion current (TIC)data was acquired over 500-4500 m/z range using MassLynx dataacquisition software (Waters Corporation). Sample component mass datawas acquired in the positive ion V-mode, and the ESI source was operatedat source temperature: 150° C., desolvation temperature: 350° C.,desolvation gas: 800 L/hr, sample cone voltage: 60 V, capillary voltage:3.0 kV, desolvation gas: nitrogen, and collision gas: argon. The summedTIC mass spectra for each peak was deconvoluted by the MaxEnt1 algorithmto generate the neutral mass data of the peak component.

Preparation of Reduced ADC Samples for UPLC/ESI-ToF MS Analysis

Reduction of the disulfide bonds in the antibody of the ADC (˜1 μg/μLsolution) to generate the light and heavy chains was performed using 20mM DTT at 60° C. for 20 minutes. An injection volume of 5-10 μL of thereduced ADC sample was employed for UPLC/ESI-ToF-MS analysis.

Exemplary ADC (PABC) for Illustration Purposes:

Note that T=Trastuzumab, which is used interchangeably with “Herceptin”herein; VC=valine-citruline; C=Cetuximab (Erbitux)

TABLE 11 ADC Cytotoxicity (EC₅₀, nM) ADC JIMT-1 NCI-N87 HCC1954*T-VC-PABC-85 — — 0.021 *T-VC-PABC-77 0.046 0.002 0.069 *T-VC-PABC-77 —— 0.023 C-VC-PABC-77 — — — *T-VC-PABC-80 — — 0.018 *T-VC-PABC-58 — —0.030 *T-VC-PABC-63 — — —

TABLE 12 ADC Cytotoxicity (EC₅₀, nM) AsPC- BxPC- HPAF PANC- ADC 1 3 II 1OE19 A549 T-VC- 0.01047 PABC-77 Cetuximab- 0.00401 0.03673 0.026570.1441 0.09405 VC-PABC-77

TABLE 13 ADC Cytotoxicity (EC₅₀, nM) ADC CAPAN-1 CAPAN-2 T-VC-PABC-772.035 — C-VC-PABC-77 — 0.115

Example 4 Efficacy Study of Toxins in PC-3 Tumour-Bearing Mice

Test articles were administered IV. Dosage was as indicated in FIG. 14,each being dosed near maximum tolerated dosage. One injection of testarticle was delivered every seven days for four repeats/injections(compound D) or one injection every seven days for threerepeats/injections (compound 23). Vehicle: 6.3% Trehalose, 0.05%Tween20, 20 mM Citrate Buffer, pH5.0, 4° C.

Procedure Overview

Thirty six (66) female athymic nude mice, purchased from HarlanLaboratories at 7-8 weeks of age, were inoculated subcutaneously in theback with 5×10⁶ PC-3 tumour cells on experimental day 0. Tumours weremeasured every Monday, Wednesday, and Friday. Once tumors reach 150-200mm³ in size (experimental day 27 to 34), animals were assigned to one of4 treatment groups by counterbalancing the average tumor size acrossgroups. Animals were treated with their respective compound asindicated, and tumour measures continued every Monday, Wednesday, andFriday. Data shows animal results to experimental day 54 or untiltumours reached 800 mm³ in size.

PC-3 Cells

Cell Preparation-Tissue Culture:

The PC-3 human prostate adenocarcinoma cell line was obtained from ATCC(Cat # CRL-1435) in 2002.

Cells were started from a frozen vial of lab stock which were frozendown from ATCC original vial, tested for mycoplasma negative and kept inlab liquid nitrogen tanks. Cell cultures with passage #3 to #10 and aconfluence of 80-90% were harvested for in vivo studies. Cells weregrown in Ham's F12 medium supplemented with 2 mM L-glutamine and 10% FBSat 37° C. in 5% CO₂ environment. Cells were sub-cultured once a weekwith split ratio 1:3 to 1:6 and expanded. The medium was renewed once aweek.

Cell Preparation—Harvesting for Implantation

Cells were rinsed briefly one time with 2 mL of fresh Trypsin/EDTAsolution (0.25% trypsin with EDTA 4Na), then the extra trypsin/EDTA wasaspirated. Then 1.5 mL of Trysin/EDTA was added, the flask was laidhorizontally to ensure the cells were covered by trypsin/EDTA. The cellswere then incubated at 37° C. for a few minutes. The cells were observedunder an inverted microscope to ensure the cell layer was dispersed,then fresh medium was added, and 50 μL of cell suspension was sampledand mixed with trypan blue (1:1) and the cells were counted and cellviability assessed using the Cellometer Auto T4. The cells werecentrifuged at 1,000 rpm for 7 min and the supernatant aspirated. Thecells were then re-suspend in growth medium to the appropriateconcentration for inoculation. Injection volume was 100 μL per animal.

Tumour Cell Implantation—SC Back

On Day 0, 5.0×10⁶ tumour cells was implanted subcutaneously into theback of mice in a volume of 100 μL using a 27/28-gauge needle underIsoflurane anesthesia.

Animal Housing

Animals were housed in ventilated cages, 2 to 5 animals per cage, in a12-hour light/dark cycle. Animals received sterile food and water adlibitum and housing and use of animals was performed in accordance withCanadian Council on Animal Care guidelines. Animals were handledaseptically, and cages changed once every 10-14 days.

Data Collection (Tumour Size)

Mice were monitored every Monday, Wednesday and Friday for tumourdevelopment. Dimensions of established tumours was measured withcalipers. Tumour volumes were calculated according to the equationL×W²/2 with the length (mm) being the longer axis of the tumour. Animalswere also weighed at the time of tumour measurement. Tumours wereallowed to grow to a maximum of 800 mm³.

Institutional Animal Care Committee

The methodology used was reviewed and approved by the University ofBritish Columbia Animal Care Committee (ACC) prior to conducting thestudies to ensure studies were planned in accordance with the CanadianCouncil on Animal Care guidelines. During the study the care, housingand use of animals was performed in accordance with the Canadian Councilon Animal Care guidelines.

Analysis Methods

Tumour Volume X Experimental Day Growth Curves

Tumour volumes of each group across the treatment days were plotted.Growth curves were cutoff for each group at the time point when thefirst animal reached the tumour-size experimental endpoint (800 mm3), orat the last day of the study. Any animal that was withdrawn from thestudy prior to the group growth curve cutoff was removed entirely fromthe study.

Animal Exclusions

Any animal with ulcerating tumours, necessitating euthanasia of theanimal, with tumour volume of 700 mm³ or smaller were removed from thestudy and did not contribute to the data analysis (except for Days toRecurrence if the final tumour volume was >2.0 fold higher than on thetreatment day).

Example 5 Efficacy Dose Range Finding of Antibody Drug Conjugates in theNCI-N87 Tumour Model Using NOD SCID Gamma Mice

Test articles were administered IV, one treatment only. “T” refers toTrastuzumab. Dosage was as indicated in FIG. 15. Vehicle: 20 mM SodiumCitrate, 6.3% Trehalose, 0.02% Tween-20, pH 5, 4° C.

Procedure Overview

Seventy six (76) female NOD/SCID Gamma mice (NSG), purchased from TheJackson Laboratory (JAX® Mice) at 7-8 weeks of age, were inoculatedsubcutaneously in the lower back with 5×10⁶ NCI-N87 tumour cells inmatrigel on experimental day 0. Tumours were measured every Monday,Wednesday, and Friday. Once tumors reach 150-200 mm³ in size(experimental day 27), animals were assigned to one of 10 treatmentgroups by counterbalancing the average tumor size across groups. Animalswere treated with their respective compound as indicated, and tumourmeasures continued every Monday, Wednesday, and Friday. Data showsanimal results to experimental day 50 or until tumours reached 800 mm³in size.

Cell Preparation-Tissue Culture

NCI-N87 Cells

NCI-N87 human gastric carcinoma cells were derived from a livermetastasis of a well differentiated carcinoma of the stomach taken priorto cytotoxic therapy. The tumor was passaged as a xenograft in athymicnude mice for three passages before the cell line was established.NCI-N87 cells were obtained under MTA from the ATCC (Cat #CRL-5822) in2013 and were tested negative at RADIL for Mycoplasma and mousepathogens. (RADIL certificate #: 10556-2013)

Cells were started from a frozen vial of lab stock which was frozen downfrom ATCC original vial and kept in lab liquid nitrogen tanks. Cellcultures with passage #3 to #10 and a confluence of 80-90% wereharvested for in vivo studies. NCI-N87 cells were grown in RPMI 1640medium with 1.0 mM L-glutamine and 10% FBS at 37° C. in 5% CO2environment. Cells were subcultured once or twice a week with the splitratio 1:3 or 1:4 and expanded. The medium was renewed once a week. Cellwere frozen with 5% DMSO.

Cell Preparation—Harvesting for Implantation

Cells were rinsed briefly one time with Hanks Balanced Salt Solutionwithout Ca, Mg. Fresh Trypsin/EDTA solution (0.25% trypsin with EDTA4Na) was added, and the flask laid horizontally to ensure the cells werecovered by trypsin/EDA, and then the extra trypsin/EDTA was aspirated.The cells were incubated at 37° C. for a few minutes. Cells wereobserved under an inverted microscope until cell layer is dispersed,fresh medium is then added. Then, 50 μL of cell suspension was collectedand mix with trypan blue (1:1) and the cells counted and assessed forviability on a haemocytometer. Viability should be ≧90%. The cells werecentrifuged at 125 RCF (1000 rpm) for 7 min and the supernatantaspirated off. The cells were resuspended in cold growth medium to 2times the desired final concentration (100×10⁶/mL). The suspension wasmixed (on ice) with matrigel (1:1). The resulting cell suspensions(50×10⁶ cells/mL) was used to deliver 5×10⁶ cells in an injection volumeof 100 μL per animal. All equipment coming into contact with matrigel(needles, syringes, pipette tips) were chilled prior to injection.

Tumour Cell Implantation—Subcutaneous (NCI-N87)

Prior to inoculation, approximately 2×2 cm area was shaved in the lowerback region of each mouse and cleaned with alcohol. On Day 0, 5.0×10⁶tumour cells were implanted subcutaneously into the back of mice in avolume of 100 μL using a 27/28-gauge needle under Isoflurane anesthesia.

Animal Housing

Animals were housed in ventilated cages, 2 to 5 animals per cage, in a12-hour light/dark cycle. Animals received sterile food and water adlibitum and housing and use of animals was performed in accordance withCanadian Council on Animal Care guidelines. Animals were handledaseptically, and cages changed once every 10-14 days.

Data Collection (Tumour Size)

Mice were monitored every Monday, Wednesday and Friday for tumourdevelopment. Dimensions of established tumours was measured withcalipers. Tumour volumes were calculated according to the equationL×W²/2 with the length (mm) being the longer axis of the tumour. Animalswere also weighed at the time of tumour measurement. Tumours wereallowed to grow to a maximum of 800 mm³.

Institutional Animal Care Committee

The methodology used was reviewed and approved by the University ofBritish Columbia Animal Care Committee (ACC) prior to conducting thestudies to ensure studies were planned in accordance with the CanadianCouncil on Animal Care guidelines. During the study the care, housingand use of animals was performed in accordance with the Canadian Councilon Animal Care guidelines.

Analysis Methods

Tumour Volume X Experimental Day Growth Curves

Tumour volumes of each group across the treatment days were plotted.Growth curves were cutoff for each group at the time point when thefirst animal reached the tumour-size experimental endpoint (800 mm3), orat the last day of the study. Any animal that was withdrawn from thestudy prior to the group growth curve cutoff was removed entirely fromthe study.

Animal Exclusions

Any animal with ulcerating tumours, necessitating euthanasia of theanimal, with tumour volume of 700 mm³ or smaller were removed from thestudy and did not contribute to the data analysis (except for Days toRecurrence if the final tumour volume was >2.0 fold higher than on thetreatment day).

Example 6 Efficacy Comparison of Antibody Drug Conjugates in the NCI-N87Tumour Model Using NOD SCID Gamma Mice

Test articles were administered IV, with one administration. Dosageswere as indicated in FIG. 16. “T” refers to Trastuzumab. Vehicle: 20 mMSodium Citrate, 6.3% Trehalose, 0.02% Tween-20, pH 5, 4° C.

Procedure Overview

Twenty-four (24) female NOD/SCID Gamma mice (NSG), purchased from TheJackson Laboratory (JAX® Mice) at 7-8 weeks of age, were inoculatedsubcutaneously in the lower back with 5×10⁶ NCI-N87 tumour cells inmatrigel on experimental day 0. Tumours were measured every Monday,Wednesday, and Friday. Once tumors reach 150-200 mm³ in size(experimental day 27), animals were assigned to one of 3 treatmentgroups by counterbalancing the average tumor size across groups. Animalswere treated with their respective compound as outlined, and tumourmeasures continued every Monday, Wednesday, and Friday. Data showsanimal results to experimental day 88 or until tumours reached 800 mm³in size.

Cell Preparation-Tissue Culture

NCI-N87 Cells

NCI-N87 human gastric carcinoma cells were derived from a livermetastasis of a well differentiated carcinoma of the stomach taken priorto cytotoxic therapy. The tumor was passaged as a xenograft in athymicnude mice for three passages before the cell line was established.NCI-N87 cells were obtained under MTA from the ATCC (Cat # CRL-5822) in2013 and were tested negative at RADIL for Mycoplasma and mousepathogens. (RADIL certificate #: 10556-2013)

Cells were started from a frozen vial of lab stock which was frozen downfrom ATCC original vial and kept in lab liquid nitrogen tanks. Cellcultures with passage #3 to #10 and a confluence of 80-90% wereharvested for in vivo studies. NCI-N87 cells were grown in RPMI 1640medium with 1.0 mM L-glutamine and 10% FBS at 37° C. in 5% CO2environment. Cells were subcultured once or twice a week with the splitratio 1:3 or 1:4 and expanded. The medium was renewed once a week. Cellwere frozen with 5% DMSO.

Cell Preparation—Harvesting for Implantation

Cells were rinsed briefly one time with Hanks Balanced Salt Solutionwithout Ca, Mg. Fresh Trypsin/EDTA solution (0.25% trypsin with EDTA4Na) was added, and the flask laid horizontally to ensure the cells werecovered by trypsin/EDA, and then the extra trypsin/EDTA was aspirated.The cells were incubated at 37° C. for a few minutes. Cells wereobserved under an inverted microscope until cell layer is dispersed,fresh medium is then added. Then, 50 μL of cell suspension was collectedand mix with trypan blue (1:1) and the cells counted and assessed forviability on a haemocytometer. Viability should be ≧90%. The cells werecentrifuged at 125 RCF (1000 rpm) for 7 min and the supernatantaspirated off. The cells were resuspended in cold growth medium to 2times the desired final concentration (100×10⁶/mL). The suspension wasmixed (on ice) with matrigel (1:1). The resulting cell suspensions(50×10⁶ cells/mL) was used to deliver 5×10⁶ cells in an injection volumeof 100 μL per animal. All equipment coming into contact with matrigel(needles, syringes, pipette tips) were chilled prior to injection.

Tumour Cell Implantation—Subcutaneous (NCI-N87)

Prior to inoculation, approximately 2×2 cm area was shaved in the lowerback region of each mouse and cleaned with alcohol. On Day 0, 5.0×10⁶tumour cells were implanted subcutaneously into the back of mice in avolume of 100 μL using a 27/28-gauge needle under Isoflurane anesthesia.

Animal Housing

Animals were housed in ventilated cages, 2 to 5 animals per cage, in a12-hour light/dark cycle. Animals received sterile food and water adlibitum and housing and use of animals was performed in accordance withCanadian Council on Animal Care guidelines. Animals were handledaseptically, and cages changed once every 10-14 days.

Data Collection (Tumour Size)

Mice were monitored every Monday, Wednesday and Friday for tumourdevelopment. Dimensions of established tumours was measured withcalipers. Tumour volumes were calculated according to the equationL×W²/2 with the length (mm) being the longer axis of the tumour. Animalswere also weighed at the time of tumour measurement. Tumours wereallowed to grow to a maximum of 800 mm³.

Institutional Animal Care Committee

The methodology used was reviewed and approved by the University ofBritish Columbia Animal Care Committee (ACC) prior to conducting thestudies to ensure studies were planned in accordance with the CanadianCouncil on Animal Care guidelines. During the study the care, housingand use of animals was performed in accordance with the Canadian Councilon Animal Care guidelines.

Analysis Methods

Tumour Volume X Experimental Day Growth Curves

Tumour volumes of each group across the treatment days were plotted.Growth curves were cutoff for each group at the time point when thefirst animal reached the tumour-size experimental endpoint (800 mm3), orat the last day of the study. Any animal that was withdrawn from thestudy prior to the group growth curve cutoff was removed entirely fromthe study.

Animal Exclusions

Any animal with ulcerating tumours, necessitating euthanasia of theanimal, with tumour volume of 700 mm³ or smaller were removed from thestudy and did not contribute to the data analysis (except for Days toRecurrence if the final tumour volume was >2.0 fold higher than on thetreatment day).

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the disclosure have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the disclosure. Accordingly, the disclosure isnot limited except as by the appended claims.

What is claimed is:
 1. A compound having the following structure (I):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: R₁ is selected from the group consisting of optionallysubstituted alkyl, optionally substituted alkylamino, optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted heterocyclyl and optionally substituted heteroaryl; R₂ isselected from the group consisting of optionally substituted alkyl,optionally substituted alkylamino, optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted heterocyclyl andoptionally substituted heteroaryl; R₃ is selected from the groupconsisting of H and C₁₋₆alkyl; R₄ is selected from the group consistingof H and C₁₋₆alkyl; and R₅ is selected from the group consisting ofC₁₋₆alkyl and —SH.
 2. The compound according to claim 1, wherein eachoptionally substituted alkyl, optionally substituted alkylamino,optionally substituted cycloalkyl, optionally substituted aryl,optionally substituted heterocyclyl and optionally substitutedheteroaryl is, independently, optionally substituted with ═O, ═S, —OH,—OR₆, —O₂CR₆, —SH, —SR₆, —SOCR₆, —NH₂, —N₃, —NHR₆, —N(R₆)₂, —NHCOR₆,—NR₆COR₆, —I, —Br, —Cl, —F, —CN, —CO₂H, —CO₂R₆, —CHO, —COR₆, —CONH₂,—CONHR₆, —CON(R₆)₂, —COSH, —COSR₆, —NO₂, —SO₃H, —SOR₆ or —SO₂R₆, whereineach R₆ is, independently, alkyl optionally substituted with halogen,—OH or —SH.
 3. The compound according to claim 1, wherein eachoptionally substituted aryl and optionally substituted heteroaryl is,independently, selected from the group consisting of optionallysubstituted phenyl, optionally substituted naphthyl, optionallysubstituted anthracyl, optionally substituted phenanthryl, optionallysubstituted furyl, optionally substituted pyrrolyl, optionallysubstituted thiophenyl, optionally substituted benzofuryl, optionallysubstituted benzothiophenyl, optionally substituted quinolinyl,optionally substituted isoquinolinyl, optionally substituted imidazolyl,optionally substituted thiazolyl, optionally substituted oxazolyl, andoptionally substituted pyridinyl.
 4. The compound according to claim 1,wherein R₂ is selected from one of the following structures (III), (IV),(V), (VI):

wherein: Q is CR₇ or N; Z is C(R₇)₂, NR₇, S, or O; wherein in structure(VI), one instance of Z is CR₇ or N, and the other instance is C(R₇)₂,NR₇, S, or O; each R₇ is, independently, selected from the groupconsisting of H, —OH, —OR₆, —O₂CR₆, —SH, —SR₆, —SOCR₆, —NH₂, —N₃, —NHR₆,—N(R₆)₂, —NHCOR₆, —NR₆COR₆, —I, —Br, —Cl, —F, —CN, —CO₂H, —CO₂R₆, —CHO,—COR₆, —CONH₂, —CONHR₆, —CON(R₆)₂, —COSH, —COSR₆, —NO₂, —SO₃H, —SOR₆ or—SO₂R₆, wherein each R₆ is, independently, alkyl optionally substitutedwith halogen, —OH or —SH.
 5. The compound according to claim 4, whereinR₂ is selected from the group consisting of:


6. The compound according to claim 5 wherein R₂ is:


7. The compound according to claim 1, wherein R₃, R₄ and R₅ are eachmethyl.
 8. The compound according to claim 1, wherein R₃ is H, R₄ ismethyl, and R₅ is methyl.
 9. The compound according to claim 1, which is(S,E)-N-(benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.10. The compound according to claim 1, which is(S,E)-2,5-dimethyl-N-(4-(2,2,2-trifluoroacetamido)phenylsulfonyl)-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.11. The compound according to claim 1, which is(S,E)-N-(4-aminophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.12. The compound according to claim 1, which is(S,E)-4-((S)-2-((S)-3-(4-(aminomethyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.13. The compound according to claim 1, which is(S,E)-N-(3-aminophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.14. The compound according to claim 1, which is(S,E)-N-(4-(1-aminocyclopropyl)benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.15. The compound according to claim 1, which is(S,E)-N-(4-(1-aminocyclopropyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.16. The compound according to claim 1, which is(S,E)-N-(4-(aminomethyl)benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.17. The compound according to claim 1, which is(S,E)-N-(4-(aminomethyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.18. A method of inhibiting tumor growth in a mammal, comprisingadministering to a mammal in need thereof an effective amount of thecompound of any one of claims 1-8 and 9-17.
 19. A pharmaceuticalcomposition comprising the compound of any one of claims 1-8 and 9-17,or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof,and a pharmaceutically acceptable carrier, diluent or excipient.
 20. Amethod of inhibiting tumor growth in a mammal, comprising administeringto a mammal in need thereof an effective amount of the pharmaceuticalcomposition of claim
 19. 21. A composition having the followingstructure:(T)-(L)-(D)   (II) wherein (T) is a targeting moiety, (L) is an optionallinker, and (D) is a compound having the following structure (I):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: R₁ is selected from the group consisting of optionallysubstituted alkyl, optionally substituted alkylamino, optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted heterocyclyl and optionally substituted heteroaryl; R₂ isselected from the group consisting of optionally substituted alkyl,optionally substituted alkylamino, optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted heterocyclyl andoptionally substituted heteroaryl; R₃ is selected from the groupconsisting of H and C₁₋₆alkyl; R₄ is selected from the group consistingof H and C₁₋₆alkyl; and R₅ is selected from the group consisting ofC₁₋₆alkyl and —SH.
 22. The composition according to claim 21, whereineach optionally substituted alkyl, optionally substituted alkylamino,optionally substituted cycloalkyl, optionally substituted aryl,optionally substituted heterocyclyl and optionally substitutedheteroaryl is, independently, optionally substituted with ═O, ═S, —OH,—OR₆, —O₂CR₆, —SH, —SR₆, —SOCR₆, —NH₂, —N₃, —NHR₆, —N(R₆)₂, —NHCOR₆,—NR₆COR₆, —I, —Br, —Cl, —F, —CN, —CO₂H, —CO₂R₆, —CHO, —COR₆, —CONH₂,—CONHR₆, —CON(R₆)₂, —COSH, —COSR₆, —NO₂, —SO₃H, —SOR₆ or —SO₂R₆, whereineach R₆ is, independently, alkyl optionally substituted with halogen,—OH or —SH.
 23. The composition according to claim 21, wherein eachoptionally substituted aryl and optionally substituted heteroaryl is,independently, selected from the group consisting of optionallysubstituted phenyl, optionally substituted naphthyl, optionallysubstituted anthracyl, optionally substituted phenanthryl, optionallysubstituted furyl, optionally substituted pyrrolyl, optionallysubstituted thiophenyl, optionally substituted benzofuryl, optionallysubstituted benzothiophenyl, optionally substituted quinolinyl,optionally substituted isoquinolinyl, optionally substituted imidazolyl,optionally substituted thiazolyl, optionally substituted oxazolyl, andoptionally substituted pyridinyl.
 24. The composition according to claim21, wherein R₂ is selected from one of the following structures (III),(IV), (V), (VI):

wherein: Q is CR₇ or N; Z is C(R₇)₂, NR₇, S, or O; wherein in structure(VI), one instance of Z is CR₇ or N, and the other instance is C(R₇)₂,NR₇, S, or O; each R₇ is, independently, selected from the groupconsisting of H, —OH, —OR₆, —O₂CR₆, —SH, —SR₆, —SOCR₆, —NH₂, —N₃, —NHR₆,—N(R₆)₂, —NHCOR₆, —NR₆COR₆, —I, —Br, —Cl, —F, —CN, —CO₂H, —CO₂R₆, —CHO,—COR₆, —CONH₂, —CONHR₆, —CON(R₆)₂, —COSH, —COSR₆, —NO₂, —SO₃H, —SOR₆ or—SO₂R₆, wherein each R₆ is, independently, alkyl optionally substitutedwith halogen, —OH or —SH.
 25. The composition according to claim 24,wherein R₂ is selected from the group consisting of:


26. The composition according to claim 25 wherein R₂ is:


27. The composition according to claim 21, wherein R₃, R₄ and R₅ areeach methyl.
 28. The composition according to claim 21, wherein R₃ is H,R₄ is methyl, and R₅ is methyl.
 29. The composition according to claim21, wherein (D) is(S,E)-N-(benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:


30. The composition according to claim 21, wherein (D) is(S,E)-N-(4-aminophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:


31. The composition according to claim 21, wherein (D) is(S,E)-4-((S)-2-((S)-3-(4-(aminomethyl)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-N-(benzylsulfonyl)-2,5-dimethylhex-2-enamide,having the following structure:


32. The composition according to claim 21, wherein (D) is(S,E)-N-(3-aminophenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:


33. The composition according to claim 21, wherein (D) is(S,E)-N-(4-(1-aminocyclopropyl)benzylsulfonyl)-2,5-dimethyl-44(S)—N,3,3-trimethyl-24(S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:


34. The composition according to claim 21, wherein (D) is(S,E)-N-(4-(1-aminocyclopropyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:


35. The composition according to claim 21, wherein (D) is(S,E)-N-(4-(aminomethyl)benzylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:


36. The composition according to claim 21, wherein (D) is(S,E)-N-(4-(aminomethyl)phenylsulfonyl)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enamide,having the following structure:


37. The composition according to claim 21, wherein (L-D) isMC-VC-PABC-77, having the following structure:


38. The composition according to claim 21, wherein (L-D) is4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl4-(N—((S,E)-2,5-dimethyl-4-((S)—N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoyl)sulfamoyl)benzylcarbamate(MC-VC-PABC-85), having the following structure:


39. The composition according to claim 21, wherein (L-D) isMC-VC-PABC-41, having the following structure:


40. The composition according to claim 21, wherein (L-D) isMC-VC-PABC-58, having the following structure:


41. The composition according to claim 21, wherein (L-D) isMC-VC-PABC-63, having the following structure:


42. The composition according to claim 21, wherein (T-L-D) ismAb-MC-VC-PABC-58, having the following structure:

wherein mAb is trastuzumab.
 43. The composition according to claim 21,wherein (T-L-D) is mAb-MC-VC-PABC-63, having the following structure:

wherein mAb is trastuzumab.
 44. A method of killing cancer cells in amammal, comprising administering to a mammal in need thereof aneffective amount of the composition of any one of claims 21-43.
 45. Amethod of inhibiting tumor growth in a mammal, comprising administeringto a mammal in need thereof an effective amount of the composition ofany one of claims 21-43.
 46. A pharmaceutical composition, comprisingthe composition of any one of claims 21-43, or a stereoisomer,pharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable carrier, diluent or excipient.
 47. A methodof inhibiting tumor growth in a mammal, comprising administering to amammal in need thereof an effective amount of the pharmaceuticalcomposition of claim
 46. 48. A method of killing cancer cells in amammal, comprising administering to a mammal in need thereof aneffective amount of the pharmaceutical composition of claim 46.